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An alpha4beta1/alpha4beta7 dual antagonist, 35S-compound 1, was used as a model ligand to study the effect of divalent cations on the activation state and ligand binding properties of alpha4 integrins. In the presence of 1 mM each Ca2+/Mg2+, 35S-compound 1 bound to several cell lines expressing both alpha4beta1 and alpha4beta7, but 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino) pentanoylamino]-butyric acid (BIO7662), a specific alpha4beta1 antagonist, completely inhibited 35S-compound 1 binding, suggesting that alpha4beta1 was responsible for the observed binding. 35S-Compound 1 bound RPMI-8866 cells expressing predominantly alpha4beta7 with a KD of 1.9 nM in the presence of 1 mM Mn2+, and binding was inhibited only 29% by BIO7662, suggesting that the probe is a potent antagonist of activated alpha4beta7. With Ca2+/Mg2+, 35S-compound 1 bound Jurkat cells expressing primarily alpha4beta1 with a KD of 18 nM. In contrast, the binding of 35S-compound 1 to Mn2+-activated Jurkat cells occurred slowly, reaching equilibrium by 60 min, and failed to dissociate within another 60 min. The ability of four alpha4beta1/alpha4beta7 antagonists to block binding of activated alpha4beta1 or alpha4beta7 to vascular cell adhesion molecule-1 or mucosal addressin cell adhesion molecule-1, respectively, or to 35S-compound 1 was measured, and a similar rank order of potency was observed for native ligand and probe. Inhibition of 35S-compound 1 binding to alpha4beta1 in Ca2+/Mg2+ was used to identify nonselective antagonists among these four. These studies demonstrate that alpha4beta1 and alpha4beta7 have distinct binding properties for the same ligand, and binding parameters are dependent on the state of integrin activation in response to different divalent cations.

Integrin alpha 4 beta 1 plays an important role in inflammatory processes by regulating the migration of lymphocytes into inflamed tissues. Here we evaluated the biochemical, pharmacological, and pharmacodynamic properties and efficacy in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, of two types of alpha 4 beta 1 inhibitors, the anti-rat alpha 4 monoclonal antibody TA-2 and the small molecule inhibitor BIO5192 [2(S)-[[1-(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino]-4-[4-methyl-2(S)-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino)-pentanoylamino]-butyric acid]. TA-2 has been extensively studied in rats and provides a benchmark for assessing function. BIO5192 is a highly selective and potent (KD of <10 pM) inhibitor of alpha 4 beta 1. Dosing regimens were identified for both inhibitors, which provided full receptor occupancy during the duration of the study. Both inhibitors induced leukocytosis, an effect that was used as a pharmacodynamic marker of activity, and both were efficacious in the EAE model. Treatment with TA-2 caused a decrease in alpha 4 integrin expression on the cell surface, which resulted from internalization of alpha 4 integrin/TA-2 complexes. In contrast, BIO5192 did not modulate cell surface alpha 4 beta 1. Our results with BIO5192 indicate that alpha 4 beta 7 does not play a role in this model and that blockade of alpha 4 beta 1/ligand interactions without down-modulation is sufficient for efficacy in rat EAE. BIO5192 is highly selective and binds with high affinity to alpha 4 beta 1 from four of four species tested. These studies demonstrate that BIO5192, a novel, potent, and selective inhibitor of alpha 4 beta 1 integrin, will be a valuable reagent for assessing alpha 4 beta 1 biology and may provide a new therapeutic for treatment of human inflammatory diseases.

A nonpeptidyl small molecule antagonist, compound A, to nonactivated very late antigen-4 (VLA4) was examined in lung inflammation induced by a single dose of ovalbumin challenge. Compound A presented a good pharmacokinetic property, when given intratracheally, and the blood cells from such pharmacokinetic study showed good receptor occupancy of the compound for approximately 8 hours. Compound A was then tested in an ovalbumin-induced airway inflammation model by intranasal or intravenous route of administration. There was a dose-dependent inhibition of eosinophilia in the bronchiolar lavage fluid, when compound A was given intranasally but not when it was given intravenously. For comparison, antibody to VLA4 and another compound, BIO1211, which reacts only with activated VLA4, were examined in this system. Immunohistochemical analyses of the lung tissue substantiated the findings in the bronchiolar lavage fluid. Specific staining of the major basic protein of eosinophils showed peribronchiolar infiltration of eosinophils. Some of these eosinophils were also positive for nitrotyrosine, suggesting activation of eosinophils in the lung interstitium. There was deposition of major basic protein and nitrotyrosine at the base of the perivascular endothelium, indicative of degranulation of eosinophils in the area. After intranasal treatment with compound A, eosinophils in the lungs and their activation products were substantially decreased, documenting its effectiveness in inhibiting lung inflammation.

We have developed a cell-free assay for binding of solubilized beta1 integrins to their physiologically relevant ligands using an electrochemiluminescent detection method. The method utilizes ruthenium-conjugated monoclonal antibodies for detection of either purified integrins or, more conveniently, integrin-expressing cell lysates, which are captured on beads coated with extracellular matrix or vascular ligand proteins. For the interaction of alpha1beta1 integrin with collagen IV, a signal of 10-fold over background was generated with samples containing only 10 ng (0.05 pmol) of integrin. This interaction is cation-dependent and can be inhibited by blocking antibodies to the alpha1 subunit. The method was extended to studies of ligand binding by integrins alpha2beta1, alpha4beta1, alpha5beta1, and alpha6beta1. For each integrin-ligand pair, the specificity of the interaction was verified with neutralizing antibodies against the specific integrin. The specific binding signal correlated with the activating ability of the labeled antibody used for detection, although the ability of divalent cations (Mn2+, Mg2+, Ca2+) to support integrin-ligand binding varied dramatically among the various integrin-ligand pairs. The assay provides a simple method for investigating integrin-ligand interactions without avidity and/or signaling effects which can complicate conventional cell-based assay methods.

Integrins alpha9beta1 and alpha4beta1 form a distinct structural class, but while alpha4beta1 has been subjected to extensive study, alpha9beta1 remains poorly characterized. We have used the small molecule N-(benzenesulfonyl)-(L)-prolyl-(L)-O-(1-pyrrolidinylcarbonyl)tyrosine (3) to investigate the biochemical properties of alpha9beta1 and directly compare these properties with those of alpha4beta1. Compound 3 has a high affinity for both integrins with K(D) values of < or =3 and 180 pM for alpha9beta1 in 1 mM Mn2+ (activating) and 1 mM Ca2+ and 1 mM Mg2+ (nonactivating) conditions and < or =5 and 730 pM for alpha4beta1 under the corresponding conditions. Ca2+ treatment promoted the binding of 3 to both integrins (EC50 = 30 microM Ca2+ in both cases). Compound 3 binding to both integrins was also stimulated by the addition of the activating monoclonal antibody TS2/16. These findings indicate that the mechanisms by which metal ions and TS2/16 regulate ligand binding to alpha9beta1 and alpha4beta1 are similar. The binding of 3 to both integrins induced the mAb 9EG7 LIBS epitope, a property consistent with occupancy of the receptor's ligand binding site by 3. But whereas EGTA treatment inhibited the binding of 9EG7 to alpha4beta1, it stimulated the binding of 9EG7 to alpha9beta1. The 9EG7 and TS2/16 effects point to contributions of the beta1-chains on binding. Cross-linking data revealed that the integrin alpha-chains are also involved in binding the small molecule, as stable linkages were observed on both the alpha9 chain of alpha9beta1 and the alpha4 chain of alpha4beta1. Extensive structure-activity analyses with natural and synthetic ligands indicate distinct features of the ligand binding pockets. Most notable was the estimated >1000-fold difference in the affinity of the integrins for VCAM-1, which binds alpha4beta1with an apparent K(D) of 10 nM and alpha9beta1 with an apparent K(D) of >10 microM. Differences were also seen in the binding of alpha9beta1 and alpha4beta1 to osteopontin. Compound 3 competed effectively for the binding of VCAM-1 and osteopontin to both integrins. While these studies show many similarities in the biochemical properties of alpha9beta1 and alpha4beta1, they identify important differences in their structure and function that can be exploited in the design of selective alpha9beta1 and alpha4beta1 inhibitors.

The therapeutic effects of the Sonic hedgehog (Shh) have been difficult to evaluate because of its relatively short serum half-life. To address this issue polyethylene glycol modification (PEGylation) was investigated as an approach to improve systemic exposure. Shh was PEGylated by a targeted approach using cysteines that were engineered into the protein by site-directed mutagenesis as the sites of attachment. Sixteen different versions of the protein containing one, two, three, or four sites of attachment were characterized. Two forms were selected for extensive testing in animals, Shh A192C, which provided a single site for PEGylation, and Shh A192C/N91C, which provided two sites. The PEGylated proteins were evaluated for reaction specificity by SDS-PAGE and peptide mapping, in vitro potency, pharmacokinetic and pharmacodynamic properties, and efficacy in a sciatic nerve injury model. Targeted PEGylation was highly selective for the engineered cysteines and had no deleterious effect on Shh function in vitro. Systemic clearance values in rats decreased from 117.4 mL/h/kg for unmodified Shh to 29.4 mL/h/kg for mono-PEGylated Shh A192C that was modified with 20 kDa PEG-maleimide and to 2.5 mL/h/kg for di-PEGylated Shh A192C/N91C modified with 2, 20 kDa PEG vinylsulfone adducts. Serum half-life increased from 1 h for unmodified Shh to 7.0 and 12.6 h for the mono- and di-PEGylated products. These changes in clearance and half-life resulted in higher serum levels of Shh in the PEG-Shh-treated animals. In Ptc-LacZ knock-in mice expressing lacZ under regulation of the Shh receptor Patched, about a 10-fold lower dose of PEG-Shh was needed to induce beta-galactosidase than for the unmodified protein. Therapeutic treatment of mice with PEG-Shh enhanced the regeneration of injured sciatic nerves. These studies demonstrate that targeted PEGylation greatly alters the pharmacokinetic and pharmacodynamic properties of Shh, resulting in a form with improved pharmaceutical properties., (Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:371-387, 2002)

We characterized the early effects of anti-very late antigen (VLA-4) and its counterligand vascular cell adhesion molecule-1 (VCAM-1) antibody therapy on T cell infiltration and apoptosis in adoptive transfer experimental autoimmune neuritis of female Lewis rats. At the peak of disease, animals were treated with anti-VCAM-1 monoclonal antibody (mAb), anti-VLA-4 mAb, or the respective isotype mAb controls 18, 12, or 6 h before perfusion. Anti-VCAM-1 led to a rapid, significant increase of apoptotic T cells in the sciatic nerve with a maximum after 6 h, preceding the significant decrease of T cell infiltration seen after 18 h. This was accompanied by a significant reduction in mRNA levels for IFN-gamma and inducible nitric oxide synthase. The results for anti-VLA-4 treatment showed a similar trend. The early increase of T cell apoptosis following disruption of VLA-4/VCAM-1 interaction may reflect a novel signaling component of proapoptotic pathways.

A subclone of the NS0 murine myeloma cell line, frequently used to produce recombinant monoclonal antibodies, was found by a transmission electron microscopy method to express a surprisingly high titer of 10(11) retroviral particles per ml of culture supernatant. Infectivity assays showed a very low infectious titer with the restricted host range expected for a murine amphotropic retrovirus. A Western blot assay for the viral capsid protein was developed to confirm the high titer values and provide a means for monitoring batch consistency and virus removal during the purification process. Mass spectrometry of several of the viral Gag proteins demonstrated that the cell line appeared to produce at least two closely related retroviruses. N-terminal sequencing of three of the Gag proteins demonstrated that these retroviruses were members of the murine leukemia retroviral family. Western blot detection with an antibody for the capsid protein gave a linear standard curve over the range of 0.1-3 ng per lane. This allows the detection of viral titers as low as 6x10(7) virions per ml without the need to concentrate the sample. The Western blot method has higher throughput and less variability than transmission electron microscopy methods and has potential for monitoring viral titer and clearance during development of manufacturing processes.

We have used the highly selective alpha(4)beta(1) inhibitor 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino)-pentanoylamino]-butyric acid (BIO7662) as a model ligand to study alpha(4)beta(1) integrin-ligand interactions on Jurkat cells. Binding of [(35)S]BIO7662 to Jurkat cells was dependent on the presence of divalent cations and could be blocked by treatment with an excess of unlabeled inhibitor or with EDTA. K(D) values for the binding of BIO7662 to Mn(2+)-activated alpha(4)beta(1) and to the nonactivated state of the integrin that exists in 1 mm Mg(2+), 1 mm Ca(2+) were <10 pm, indicating that it has a high affinity for both activated and nonactivated integrin. No binding was observed on alpha(4)beta(1) negative cells. Through an analysis of the metal ion dependences of ligand binding, several unexpected findings about alpha(4)beta(1) function were made. First, we observed that Ca(2+) binding to alpha(4)beta(1) was stimulated by the addition of BIO7662. From solution binding studies on purified alpha(4)beta(1), two types of Ca(2+)-binding sites were identified, one dependent upon and the other independent of BIO7662 binding. Second, we observed that the metal ion dependence of ligand binding was affected by the affinity of the ligand for alpha(4)beta(1). ED(50) values for the metal ion dependence of the binding of BIO7762 and the binding of a lower affinity ligand, BIO1211, differed by 2-fold for Mn(2+), 30-fold for Mg(2+), and >1000-fold for Ca(2+). Low Ca(2+) (ED(50) = 5-10 microm) stimulated the binding of BIO7662 to alpha(4)beta(1). The effects of microm Ca(2+) closely resembled the effects of Mn(2+) on alpha(4)beta(1) function. Third, we observed that the rate of BIO7662 binding was dependent on the metal ion concentration and that the ED(50) for the metal ion dependence of BIO7662 binding was affected by the concentration of the BIO7662. These studies point to an even more complex interplay between metal ion and ligand binding than previously appreciated and provide evidence for a three-component coupled equilibrium model for metal ion-dependent binding of ligands to alpha(4)beta(1).

Interferon therapies suffer from a relatively short half-life of the products in circulation. To address this issue we investigated the effects of polyethylene glycol modification (PEGylation) on the pharmacokinetic properties of human interferon (IFN)-beta-1a. PEGylation with a linear 20-kDa PEG targeted at a single site on the N-terminal amine had no deleterious effect on its specific activity in an in vitro antiviral assay. In monkeys, PEG IFN-beta-1a treatment induced neopterin and beta2-microglobulin expression (pharmacodynamic markers of activity). Systemic clearance values in monkeys, rats, and mice decreased, respectively, from 232, 261, and 247 ml/h/kg for the unmodified IFN-beta-1a to 30.5, 19.2, and 18.7 ml/h/kg for the PEGylated form, while volume of distribution values decreased from 427, 280, and 328 ml/kg to 284, 173, and 150 ml/kg. The decreased clearance and volume of distribution resulted in higher serum antiviral activity in the PEG IFN-beta-1a-treated animals. In the rat, a more extensive set of dosing routes was investigated, including intraperitoneal, intratracheal, and oral administration. Bioavailability for the PEG IFN-beta-1a was similar to the unmodified protein for each of the extravascular routes examined. For the intraperitoneal route, bioavailability was almost 100%, whereas for the oral and intratracheal routes absorption was low (<5%). In rats, subcutaneous bioavailability was moderate (28%), whereas in monkeys it was approximately 100%. In all instances an improved pharmacokinetic profile for the PEGylated IFN-beta-1a was observed. These findings demonstrate that PEGylation greatly alters the pharmacokinetic properties of IFN-beta-1a, resulting in an increase in systemic exposure following diverse routes of administration.

Activation of the RET receptor tyrosine kinase by glial-derived neurotrophic factor family members is dependent on a family of coreceptors, GFRalpha1-4. GFRalpha3 preferentially binds the newest member of the glial-derived neurotrophic factor family of ligands, artemin. The major site of GFRalpha3 expression is in the dorsal root ganglion; however, the class of sensory neurons that expresses GFRalpha3 has not been reported previously. Using immunohistochemical methods, we show that the majority of dorsal root ganglion cells that express GFRalpha3 also express vanilloid receptor type 1, peripherin, RET, trkA and calcitonin gene-related peptide. In addition, a significant subpopulation of GFRalpha3-expressing cells also binds the lectin IB4. We demonstrate that GFRalpha3 artemin neurons are immunopositive for markers expected of nociceptors and include a subset of neurons distinct from the GDNF-responsive population. Our results indicate artemin may exert selective effects on pain sensation.

In circulating lymphocytes, the VLA-4 integrin preexists in multiple affinity states that mediate spontaneous tethering, rolling, and arrest on its endothelial ligand, vascular cell adhesion molecule-1 (VCAM-1). The regulation and function of VLA-4 affinity in lymphocytes has never been elucidated. We show here that p56(lck), the major Src kinase in T cells, is a key regulator of high affinity VLA-4. This high affinity is essential for the rapid development of firm adhesion of resting T cells to VCAM-1 and to their extracellular matrix ligand, fibronectin. Lck-regulated VLA-4 function does not require intact TCR nor several key components of the TCR signaling pathway, including ZAP-70 and SLP-76. Furthermore, stimulation of p56(lck) by the phosphatase inhibitor, pervanadate, triggers firm VLA-4-dependent adhesion to VCAM-1. Although Lck is not required for chemokine receptor signaling to mitogen-activated protein kinase, the presence of Lck-regulated high affinity VLA-4 also facilitates firm adhesion triggered by the chemokine, SDF-1, at short-lived contacts. Surprisingly, bond formation rates, ability to tether cells to VLA-4 ligand, and VLA-4 tether bond stability under shear flow are not affected by VLA-4 affinity or Lck activity. Thus, the ability of high affinity VLA-4 to arrest cells on VCAM-1 under flow arises from instantaneous post-ligand strengthening rather than from increased kinetic stability of individual VLA-4 bonds. These results suggest that p56(lck) maintains high affinity VLA-4 on circulating lymphocytes, which determines their ability to strengthen VLA-4 adhesion and rapidly respond to proadhesive chemokine signals at endothelial sites.

Post-translational modifications of the developmental signaling protein Sonic hedgehog (Shh) by a long-chain fatty acid at the N-terminus and cholesterol at the C-terminus greatly activate the protein in a cell-based signaling assay. To investigate the structural determinants of this activation phenomenon, hydrophobic and hydrophilic moieties have been introduced by chemical and mutagenic methods to the soluble N-terminal signaling domain of Shh and tested in both in vitro and in vivo assays. A wide variety of hydrophobic modifications increased the potency of Shh when added at the N-terminus of the protein, ranging from long-chain fatty acids to hydrophobic amino acids, with EC(50) values from 99 nM for the unmodified protein to 0.6 nM for the myristoylated form. The N-myristoylated Shh was as active as the natural form having both N- and C-terminal modifications. The degree of activation appears to correlate with the hydrophobicity of the modification rather than any specific chemical feature of the adduct; moreover, substitution with hydrophilic moieties decreased activity. Hydrophobic modifications at the C-terminus of Shh resulted in only a 2-3-fold increase in activity, and no activation was found with hydrophobic modification at other surface positions. The N-terminal modifications did not appear to alter the binding affinity of the Shh protein for the transfected receptor protein, Patched, and had no apparent effect on structure as measured by circular dichroism, thermal denaturation, and size determination. Activation of Desert Hh through modification of its N-terminus was also observed, suggesting that this is a common feature of Hh proteins.

We have defined regions in the Sonic hedgehog (Shh) molecule that are important for Patched (Ptc) receptor binding by targeting selected surface amino acid residues with probes of diverse sizes and shapes and assessing the effects of these modifications on function. Eleven amino acid residues that surround the surface of the protein were chosen for these studies and mutated to cysteine residues. These cysteines were then selectively modified with thiol-specific probes, and the modified proteins were tested for hedgehog receptor binding activity and their ability to induce differentiation of C3H10T1/2 cells into osteoblasts. Based on these analyses, approximately one-third of the Shh surface can be modified without effect on function regardless of the size of the attachment. These sites are located near to where the C terminus protrudes from the surface of the protein. All other sites were sensitive to modification, indicating that the interaction of Shh with its primary receptor Ptc is mediated over a large surface of the Shh protein. For sites Asn-50 and Ser-156, function was lost with the smallest of the probes tested, indicating that these residues are in close proximity to the Ptc-binding site. The epitope for the neutralizing mAb 5E1 mapped to a close but distinct region of the structure. The structure-activity data provide a unique view of the interactions between Shh and Ptc that is not readily attainable by conventional mapping strategies.

Background: The alpha4beta1 (or very late antigen-4 [VLA-4]) integrin is thought to play a role in inflammatory processes, mediating mononuclear leukocyte infiltration. The adventitial response to balloon injury is an important determinant of neointimal formation and arterial remodelling., Objectives: To determine whether the monoclonal antibody hHP1/2 directed against the human alpha4-integrin subunit decreases neoadventitial formation and subsequent luminal narrowing following balloon injury., Design: Randomized, double-blind, placebo controlled study., Setting: Tertiary care, Canadian university hospital vascular biology laboratory., Animals and Methods: In 16 pigs, two coronary arteries were injured with an oversized balloon, while a third coronary artery was designated as an uninjured control vessel. One hour before balloon injury, 5 mg/kg of hHP1/2 was administered to eight animals, while another eight animals received an infusion of a saline placebo. Animals were killed three and 14 days following balloon injury., Main Results: Administration of hHP1/2 resulted in an immediate decrease in circulating monocyte and lymphocyte counts. These parameters returned to normal within three days. There was a decrease in neoadventitial formation 14 days after arterial injury in pigs treated with hHP1/2 compared with controls (2.26+/-0.77 versus 3.42+/-1.01 mm, respectively, P=0.04). There was a loss of lumen area between days 3 (4.33+/-1.09 mm2) and 14 (3.09+/-0.38 mm2, P=0.02) after balloon injury in pigs treated with saline, but not in the pigs treated with hHP1/2., Conclusions: Administration of an antibody to the alpha4-integrin subunit is associated with less neoadventitial formation and less lumenal narrowing after balloon injury. This novel therapy may play an important role in modulating arterial remodelling and thereby may reduce restenosis following percutaneous coronary interventions in humans.

Lipocortin-1 exerts a potent immunosuppressive effect on pathogenic T cells. In multiple sclerosis and experimental autoimmune encephalomyelitis levels of lipocortins are raised, suggesting their involvement in the recovery from an immunological insult or in neural regeneration. To further understand the role of lipocortins in the peripheral nervous system we have characterized lipocortin-1 levels and cellular distribution of lipocortin-1 immunoreactivity in sciatic nerves of rats with experimental autoimmune neuritis (EAN), a model of human Guillain-Barré syndrome. EAN was induced actively by immunization with bovine peripheral myelin (active EAN) or by adoptive-transfer (AT-EAN) of P2-specific T cells. Cellular infiltrates in serial and semithin cryosections were characterized by immunohistochemistry. In parallel, lipocortin-1 levels in tissue extracts were quantified by a sandwich-ELISA. Only weak lipocortin-1 immunoreactivity was found in nerves of control animals injected with non-pathogenic T cells. The majority of macrophages and lymphocytes in EAN lesions exhibited lipocortin-1 immunoreactivity. Some very heavily stained cells showed a distribution and morphology similar to ED-2-positive macrophages which were abundant during early stages of EAN. Lipocortin-1 expression in T cells and macrophages was proven by immunocytochemical studies in semithin serial sections. In tissue extracts, lipocortin-1 levels increased from 0.24 +/- 0.14 micrograms/mg protein in controls receiving non-pathogenic T cells to a maximum of 0.55 +/- 0.1 micrograms/mg protein in AT-EAN at the peak of disease, and then slowly decreased during clinical recovery but still remained elevated. In dose-response studies in AT-EAN, highest values of lipocortin-1 (0. 71 +/- 0.23 micrograms/mg protein) were recorded after transfer of 2 x 10(7) T cells. Increased levels of lipocortin-1 were also measured in active EAN but occurred during the recovery phase (0.65 +/- 0.27 micrograms/mg protein). By analogy with other immune-mediated disorders, increased lipocortin-1 expression in the inflamed sciatic nerve in EAN may exert immunoregulatory functions in-situ and contribute to the termination of the autoimmune response.

During development, sonic hedgehog functions as a morphogen in both a short-range contact-dependent and in a long-range diffusable mode. Here, we show using a panel of sonic hedgehog variants that regions near the N terminus of the protein play a critical role in modulating these functions. In the hedgehog responsive cell line C3H10T1/2, we discovered that not only were some N-terminally truncated variants inactive at eliciting a hedgehog-dependent response, but they competed with the wild-type protein for function and therefore served as functional antagonists. These variants were indistinguishable from wild-type sonic hedgehog in their ability to bind the receptor patched-1, but failed to induce the hedgehog-responsive markers, Gli-1 and Ptc-1, and failed to promote hedgehog-dependent differentiation of the cell line. They also failed to support the adhesion of C3H10T1/2 cells to hedgehog-coated plates under conditions where wild-type sonic hedgehog supported binding. Structure-activity data indicated that the N-terminal cysteine plays a key regulatory role in modulating hedgehog activity. The ability to dissect patched-1 binding from signaling events in C3H10T1/2 cells suggests the presence of unidentified factors that contribute to hedgehog responses.

Recent structural and functional analyses of alpha integrin subunit I domains implicate a region in cation and ligand binding referred to as the metal ion-dependent adhesion site (MIDAS). Although the molecular interactions between Mn2+ and Mg2+ and the MIDAS region have been defined by crystallographic analyses, the role of cation in I domain function is not well understood. Recombinant alpha 1 beta 1 integrin I domain (alpha1-I domain) binds collagen in a cation-dependent manner. We have generated and characterized a panel of antibodies directed against the alpha1-I domain, and selected one (AJH10) that blocks alpha 1 beta 1 integrin function for further study. The epitope of AJH10 was localized within the loop between the alpha 3 and alpha 4 helices which contributes one of the metal coordination sites of the MIDAS structure. Kinetic analyses of antibody binding to the I domain demonstrate that divalent cation is required to stabilize the epitope. Denaturation experiments demonstrate that cation has a dramatic effect on the stabilization of the I domain structure. Mn2+ shifts the point at which the I domain denatures from 3.4 to 6.3 M urea in the presence of the denaturant, and from 49.5 to 58.6 degrees C following thermal denaturation. The structural stability provided to the alpha1-I domain by divalent cations may contribute to augmented ligand binding that occurs in the presence of these cations.

The alpha1beta1 integrin is a major cell surface receptor for collagen. Ligand binding is mediated, in part, through a 200 amino acid inserted 'I'-domain contained in the extracellular part of the integrin alpha chain. Integrin I-domains contain a divalent cation binding (MIDAS) site and require cations to interact with integrin ligands. We have determined the crystal structure of recombinant I-domain from the rat alpha1beta1 integrin at 2.2 A resolution in the absence of divalent cations. The alpha1 I-domain adopts the dinucleotide binding fold that is characteristic of all I-domain structures that have been solved to date and has a structure very similar to that of the closely related alpha2beta1 I-domain which also mediates collagen binding. A unique feature of the alpha1 I-domain crystal structure is that the MIDAS site is occupied by an arginine side chain from another I-domain molecule in the crystal, in place of a metal ion. This interaction supports a proposed model for ligand-induced displacement of metal ions. Circular dichroism spectra determined in the presence of Ca2+, Mg2+ and Mn2+ indicate that no changes in the structure of the I-domain occur upon metal ion binding in solution. Metal ion binding induces small changes in UV absorption spectra, indicating a change in the polarity of the MIDAS site environment.

We have used the highly specific alpha4beta1 inhibitor 4-((N'-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline (BIO1211) as a model LDV-containing ligand to study alpha4beta1 integrin-ligand interactions on Jurkat cells under diverse conditions that affect the activation state of alpha4beta1. Observed KD values for BIO1211 binding ranged from a value of 20-40 nM in the non-activated state of the integrin that exists in 1 mM Mg2+, 1 mM Ca2+ to 100 pM in the activated state seen in 2 mM Mn2+ to 18 pM when binding was measured after co-activation by 2 mM Mn2+ plus 10 microgram/ml of the integrin-activating monoclonal antibody TS2/16. The large range in KD values was governed almost exclusively by differences in the dissociation rates of the integrin-BIO1211 complex, which ranged from 0.17 x 10(-4) s-1 to >140 x 10(-4) s-1. Association rate constants varied only slightly under the same conditions, all falling in the narrow range from 0.9 to 2.7 x 10(6) M-1 s-1. The further increase in affinity observed upon co-activation by divalent cations and TS2/16 compared with that observed at saturating concentrations of metal ions or TS2/16 alone indicates that the mechanism by which these factors bring about activation are distinct and identified a previously unrecognized high affinity state on alpha4beta1 that had not been detected by conventional assay methods. Similar changes in affinity were observed when the binding properties of vascular cell adhesion molecule-1 and CS1 to alpha4beta1 were studied, indicating that the different affinity states detected with BIO1211 are an inherent property of the integrin.

Integrin alpha4beta1 mediates leukocyte recruitment, activation, mediator release, and apoptosis inhibition, and it plays a central role in inflammatory pathophysiology. High-affinity, selective inhibitors of alpha4beta1, based on the Leu-Asp-Val (LDV) sequence from the alternatively spliced connecting segment-1 (CS-1) peptide of cellular fibronectin, are described that employ a novel N-terminal peptide "cap" strategy. One inhibitor, BIO-1211, was approximately 10(6)-fold more potent than the starting peptide and exhibited tight-binding properties (koff = 1.4 x 10(-4) s-1, KD = 70 pM), a remarkable finding for a noncovalent, small-molecule inhibitor of a protein receptor. BIO-1211 was also 200-fold selective for the activated form of alpha4beta1, and it stimulated expression of ligand-induced epitopes on the integrin beta1 subunit, a property consistent with occupancy of the receptor's ligand-binding site. Pretreatment of allergic sheep with a 3-mg nebulized dose of BIO-1211 inhibited early and late airway responses following antigen challenge and prevented development of nonspecific airway hyperresponsiveness to carbachol. These results show that highly selective and potent small-molecule antagonists can be identified to integrins with primary specificity for peptide domains other than Arg-Gly-Asp (RGD); they confirm the generality of integrins as small molecule targets; and they validate alpha4beta1 as a therapeutic target for asthma.

We have investigated the functional role of very late antigen-4 [VLA-4 (alpha4/beta1) integrin] and vascular cell adhesion molecule-1 (VCAM-1) in experimental autoimmune neuritis (EAN), an animal model of the Guillain-Barré syndrome, using neutralizing monoclonal antibodies (mAbs) as probes. Disease was induced by intravenous adoptive transfer of P2 specific T cells (AT-EAN), or by immunization with bovine myelin (active EAN). Preventive treatment with 500 microg anti-VLA-4 mAb (TA-2) or with an isotype control antibody was given in AT-EAN 4 h before cell transfer and at day 3. Intravenous injection of 500 microg anti-VCAM-1 mAb (5F10) or a corresponding isotype control was given in AT-EAN 4 h before disease induction, and at days 2, 4 and 6. Preventive treatment of active EAN with mAb to VLA-4 or VCAM-1 was performed at days 5, 9 and 13. On immunohistochemical examination, VCAM-1 in sciatic nerve was found to be upregulated at early stages of EAN (days 3-5 after T-cell transfer), whilst no expression was noted in healthy controls. In both EAN models, blockade of VLA-4 markedly attenuated disease severity. Blockade of VCAM-1 also significantly ameliorated the disease course and diminished T-cell infiltration in sciatic nerve, but to a lesser degree. These experiments demonstrate the critical role of VLA-4 in the pathogenesis of EAN and show that upregulation of VCAM-1 expression contributes, at least in part, to the progression of the disease in the early stages. Future studies are needed to assess the potential contribution of other VLA-4 ligands.

We have used chemical cross-linking to identify sequences in integrin alpha4beta1 that are involved in its interactions with ligands. A recently described leucine-aspartic acid-valine (LDV)-based small molecule inhibitor of alpha4beta1 (BIO-1494), that contained a single reactive amino group for targeting the cross-linking, was used for these studies. The specificity of the interaction was defined by (i) the ability to block the interaction with a competitive inhibitor lacking the reactive group, (ii) the absolute requirement of divalent cations for cross-linking, and (iii) the lack of cross-linking to the functionally related integrin alpha4beta7. With ANB-NOS as the cross-linker, only the beta1 chain was labeled with BIO-1494, while with the more flexible cross-linker DSS both the alpha4 and beta1 chains were modified. Similar results were obtained when cross-linking was performed on K562 cells expressing alpha4beta1 but not on K562 cells expressing alpha2beta1. The site of cross-linking on the beta1 chain was localized by CNBr peptide mapping within residues 130-146, a region that contains the putative metal binding site DXSXS and for which analogous data had been generated with RGD binding to integrin alphaIIbbeta3. The striking similarity between the data we generated for an LDV ligand and published data for the RGD family supports the notion of a common ligand binding pocket formed by both integrin chains. The cross-linking strategy developed here should serve as a useful tool for studying alpha4beta1 function.

During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24-197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH2 terminus of the protein on the alpha-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog showed about a 30-fold increase in potency over unmodified soluble hedgehog in a cell- based (C3H10T1/2 alkaline phosphatase induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein.

Purpose: Two recombinant IFN-beta products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN-beta-1a) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN-beta-1b). The structural basis for activity differences between IFN-beta-1a and IFN-beta-1b, is determined., Methods: In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN-beta products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences., Results: IFN-beta-1a was 10 times more active than IFN-beta-1b with specific activities in a standard antiviral assay of 20 x 10(7) IU/mg for IFN-beta-1a and 2 x 10(7) IU/mg for IFN-beta-1b. Of the known structural differences between IFN-beta-1a and IFN-beta-1b, only glycosylation affected in vitro activity. Deglycosylation of IFN-beta-1a produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN-beta-1b revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 x 10(7) IU/mg). Crystallographic data for IFN-beta-1a revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure., Conclusions: Together these results suggest that the greater biological activity of IFN-beta-1a is due to a stabilizing effect of the carbohydrate on structure.

Glial cell line-derived neurotrophic factor (GDNF)-dependent activation of the tyrosine kinase receptor RET is necessary for kidney and enteric neuron development, and mutations in RET are associated with human diseases. Activation of RET by GDNF has been shown to require an accessory component, GDNFR-alpha (RETL1). We report the isolation and characterization of rat and human cDNAs for a novel cell-surface associated accessory protein, RETL2, that shares 49% identity with RETL1. Both RETL1 and RETL2 can mediate GDNF dependent phosphorylation of RET, but they exhibit different patterns of expression in fetal and adult tissues. The most striking differences in expression observed were in the adult central and peripheral nervous systems. In addition, the mechanisms by which the two accessory proteins facilitate the activation of RET by GDNF are quite distinct. In vitro binding experiments with soluble forms of RET, RETL1 and RETL2 demonstrate that while RETL1 binds GDNF tightly to form a membrane-associated complex which can then interact with RET, RETL2 only forms a high affinity complex with GDNF in the presence of RET. This strong RET dependence of the binding of RETL2 to GDNF was confirmed by FACS analysis on RETL1 and RETL2 expressing cells. Together with the recent discovery of a GDNF related protein, neurturin, these data raise the possibility that RETL1 and RETL2 have distinctive roles during development and in the nervous system of the adult. RETL1 and RETL2 represent new candidate susceptibility genes and/or modifier loci for RET-associated diseases.

We have compared a series of molecular constructs that contain the minimal DNA-binding and dimerization domain of bovine papillomavirus type 1 (BPV-1) E2 alone or this binding domain plus the adjacent 16 or 40 amino acids to test the role of the flanking sequences in E2 function. The presence of these sequences resulted in an up to eightfold increase in the affinity of E2 for its target DNA and stabilized the protein against denaturation both in the absence of DNA and in the form of DNA-protein complexes. In addition, an aspartic acid-to-tyrosine mutation within the flanking region blocked DNA binding and function. These data demonstrate that sequences flanking the core domain contribute to E2 function and are, in fact, an integral part of the DNA-binding domain of BPV-1 E2.

Evidence for a central role for the integrins alpha 4 beta 1 and alpha 4 beta 7 in leukocyte pathophysiology is rapidly accumulating. Five distinct alpha 4 mAbs, each able to block alpha 4-dependent adhesion in vitro, show beneficial effects in vivo in six different species, and in a wide variety of organ systems, including colon, lung, skin, neural tissue, pancreas, peritoneum, and the vessel wall. In particular, a clear role for these integrins in lung pathophysiology is implied on the basis of in vivo studies in four different species. Although several issues remain to be resolved, including the relative importance of alpha 4 beta 1 and alpha 4 beta 7, and the relative roles of their counterligands, VCAM1, fibronectin, and MAdCAM, the data argue that alpha 4 integrins will likely be critical to both the normal physiology and pathology of the lung in man. To this end, we (Adams, Lin, Lobb, and Gill, unpublished data) and others have generated peptidomimetic small molecule antagonists of VLA4 based on the connecting segment 1 (CS1) peptide sequence of fibronectin that are potent blockers of integrin adhesive function in vitro and show efficacy in vivo. We have found that our inhibitors are excellent blockers of both murine contact hypersensitivity, and of the LPR and AHR in the sheep allergic airways model (Abraham, Lobb, Adams, and Gill, unpublished data), and are therefore possible candidates for clinical intervention in human asthma. The use of the VCAM-Ig fusion protein as a probe for high-affinity alpha 4 integrins has further enhanced our understanding of alpha 4 integrin function in the lung. While integrin upregulation in vitro has been observed many times, and high affinity (as opposed to avidity) of integrins seen in vitro in several systems, in vivo proof of integrin upregulation to a high-affinity state has been difficult to obtain in the absence of selective probes. Our data provide key information in this regard and strongly argue not only that integrin upregulation does indeed occur in vivo, but also that it is in fact obligatory for the leukocyte pathologies we have examined to date. Further studies are clearly warranted to further examine mechanisms of action, and to confirm and extend these studies, both with the alpha 4 integrins and with other integrin families. In summary, our studies of alpha 4 integrins continue to provide novel insights into the pathophysiology of integrin function and into future directions for drug discovery.

Increased levels of lipocortins occur in the nervous system in multiple sclerosis, in experimental autoimmune encephalomyelitis and experimental neuritis at the height of disease and decrease thereafter, suggesting their potential involvement in recovery from disease. We therefore investigated whether lipocortins may suppress activation of autoimmune T cells. Antigen-specific and growth factor-mediated proliferation of T cell lines reactive with myelin basic protein (MBP) was measured in the presence of recombinant lipocortin-1, -2, and -5, and natural bovine lipocortin-1 using various concentrations and incubation periods. We also employed an N-terminal lipocortin-1 peptide spanning aa 1-26, a proteolytic fragment of lipocortin-1 where the respective N-terminal region was clipped off, tested blocking with a neutralizing antibody, and investigated the effect of alkaline phosphatase treatment. Both human recombinant and bovine lipocortin-1 had a marked suppressive effect on T cell activation by MBP and the respective immunogenic peptide. When added at 3 micrograms/ml we observed up to 90% inhibition of T cell proliferation between day 2 and 3, but not at earlier time points of activation. The inhibitory effect of human lipocortin-1 was blocked after addition of a neutralizing antibody directed against lipocortin-1. Lipocortin-2 and -5, and the N-terminal peptide of lipocortin-1 were ineffective, whereas the fragment spanning residues 27-345 of lipocortin-1 retained full activity. Treatment of bovine lipocortin-1 with alkaline phosphatase did not alter immunosuppressive properties.

Gelsolin is a widely distributed actin binding protein that regulates actin filament length. It exists in both an intracellular and an extracellular form that is derived from a single gene by alternative splicing. Both forms contain the six homologous domains that are responsible for function. Little is known regarding differences between the forms. We have used a combination of cysteine-specific modification with 4-vinylpyridine, HPLC peptide mapping methods, and mass spectrometry to analyze the disulfide structures of human plasma and cytoplasmic gelsolin. Of the five Cys residues in the human gelsolin sequence, all were present in the free thiol form in human cytoplasmic gelsolin, while only three of them were free thiols in the human plasma form. Cys residues 188 and 201 in domain 2 of plasma gelsolin were disulfide linked. Recombinant human plasma gelsolin that had been expressed intracellularly in Escherichia coli and as a secreted protein from Cos green monkey cells was also investigated. The E. coli product lacked the disulfide but could be converted to the plasma-like structure with mild oxidation while the mammalian product formed the correct disulfide prior to isolation. Structural differences were also detected by limited proteolysis with plasmin. The differences in proteolytic susceptibility were also due to perturbations in domain 2. These studies demonstrate that the intracellular and extracellular gelsolins are structurally distinct and suggest that at least some of the preparations of recombinant gelsolin that are being used to study structure/function may be improperly folded. The experiments also demonstrate a general method for the location of disulfide bonds in proteins.

We have used electrospray ionization-mass spectrometry to investigate Gag protein structure and processing in Rous sarcoma virus, the prototype of the avian sarcoma and leukemia viruses. Molecular masses determined for the mature virion proteins MA, CA, NC, and PR agree closely with those predicted by currently accepted models for their structures. However, the data for p10 imply that only about 10% of the product has the predicted mass while the remainder is missing the C-terminal methionine residue. Molecular masses also were obtained for products generated by PR cleavage in vitro of a Gag precursor polyprotein expressed in Escherichia coli. The data confirm the predicted Gag cleavage sites for PR. Thus, carboxypeptidase activity appears to be responsible for generating the des-Met form of p10. The same activity may account for the small amount of the mature des-Met CA, as previously reported. Analysis of cleavage products generated in vitro also serves to define the PR processing site separating the p2a and p2b peptides, Asn-164-Cys-165. In conjunction with published characterizations of these two peptides processed from the segment of Gag between MA and p10, these data suggest trimming of p2b by an aminopeptidase. Finally, the molecular masses determined for the MA-related species p19f, p23, and p35 now accurately define the structures of these proteins.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the alpha 4 integrins alpha 4 beta 1 (very late antigen 4: VLA4) and alpha 4 beta 7, which are constitutively expressed on many leukocyte subsets and play a key role in cell trafficking and activation. Using a recombinant VCAM-IgG fusion protein (VCAM-Ig) as a soluble ligand for alpha 4 beta 1 we directly demonstrated by fluorescence analysis that the alpha 4 beta 1 receptor can exist in different affinity states on the cell surface, and that a high affinity state is induced by manganese ions or certain activating anti-beta 1 monoclonal antibodies (Jakubowski et al., 1995b). Here we have extended these observations by developing a rapid and reproducible assay using alkaline phosphatase (AP)-coupled VCAM-Ig (VCAM-Ig-AP) which measures the interaction between VCAM1 and alpha 4 integrins in a microtiter plate format. This assay has allowed us to evaluate directly the effects of metal ions, anti-beta 1 mAbs, and different cell types and species on the VCAM1/alpha 4 integrin interaction. Most importantly, the assay system provides a means to rapidly evaluate alpha 4 integrin-directed inhibitors without the complication of post-ligand binding events inherent in adhesion assays.

The CA (capsid) protein of avian sarcoma and leukemia viruses occurs in multiple species. Only one form has been previously characterized biochemically. We have now determined that the mature CA protein of avian sarcoma and leukemia viruses exists as three species with different C termini, ending in amino acid residues A-476, A-478, and M-479 of the Gag precursor, respectively. These structures were deduced from a combination of cyanogen bromide peptide mapping, sequence analysis of tryptic peptides, and electrospray mass spectrometry. The three forms of CA were detected in the same ratios in Rous sarcoma virus and avian myeloblastosis virus and therefore are likely to represent a common feature of members of this genus of avian retroviruses. The only previously reported CA species, CAM-479, accounts for only about 36% of the total CA protein, while CAA-476 and CAA-478 account for 55 and 9%, respectively. From the analysis of peptides cleaved in vitro by PR, the viral protease, we infer that the cleavage site between A-476 and A-477 not only is recognized by PR but is the preferred site. We were unable to determine if A-478/A-479 is a cleavage site for PR or alternatively if CAA-478 results from further processing of CAM-479 by a carboxypeptidase. To study the biological significance of residues A-477 to M-479, we constructed genetically altered viruses in which deletions removed either residues 477 to 479 or 477 to 488. The resulting virus particles appeared to assembly with normal efficiencies, but the latter mutant showed slowed proteolytic processing. Neither of the mutants was infectious.

The alpha 4 beta 1-integrin (CD49d, CD29) constitutively expressed on leukocytes regulates cell migration to inflammatory sites, cell activation, and development through its interactions with two alternate ligands, vascular cell adhesion molecule-1 (VCAM-1; CD106) expressed on cytokine-activated endothelium, dendritic and stromal cells, and the extracellular matrix protein fibronectin. Another alpha 4-integrin receptor, alpha 4 beta 7, expressed on leukocytes also binds VCAM-1 and fibronectin (FN), and controls homing to mucosal tissues through its interactions with mucosal vascular addressin MAdCAM-1. In vitro studies have shown that alpha 4-dependent cell adhesion is regulated by the activation state of the cell and by divalent cations. However, the existence and role of cells with different alpha 4 activation states in vivo have not been defined. Herein we show that a soluble ligand with the two N-terminal domains of human VCAM-1 fused to a human IgG1 constant region, VCAM-Ig, binds selectively to activated alpha 4-receptors on murine cells, such as those induced by Mn2+ in vitro. To determine whether the cells identified by VCAM-Ig were required under physiologic conditions, we assessed its anti-inflammatory effect. We show that VCAM-Ig is not bound to the majority of murine alpha 4+ cells after in vivo administration, yet it significantly delays the onset of adoptively transferred autoimmune diabetes. Thus, soluble VCAM-Ig can modify alpha 4-dependent disease progression, apparently by its selective action on cells with activated alpha 4-integrin receptors, thereby providing evidence for distinct alpha 4 activation states in vivo.

Vascular cell adhesion molecule 1 (VCAM-1) represents a structurally and functionally distinct class of immunoglobulin superfamily molecules that bind leukocyte integrins and are involved in inflammatory and immune functions. X-ray crystallography defines the three-dimensional structure of the N-terminal two-domain fragment that participates in ligand binding. Residues in domain 1 important for ligand binding reside in the C-D loop, which projects markedly from one face of the molecule near the contact between domains 1 and 2. A cyclic peptide that mimics this loop inhibits binding of alpha 4 beta 1 integrin-bearing cells to VCAM-1. These data demonstrate how crystallographic structural information can be used to design a small molecule inhibitor of biological function.

Papillomaviruses are the causative agents of benign and malignant epithelial tumors of the skin and mucosa. They encode a DNA-binding protein, E2, that regulates viral transcription and replication, making it an important therapeutic target. By deleting the amino-terminal trans-activation domain of human papillomavirus type 16 (HPV-16) E2 while retaining its carboxy-terminal DNA binding and dimerization domain, an E2 repressor (E2R) that efficiently inhibits transcriptional activation by full-length HPV E2 was generated. To deliver this repressor protein into animal cells, we have utilized the human immunodeficiency virus type 1 (HIV-1) Tat protein which itself is taken up efficiently into intact cells. Chimeras of E2R and the cellular uptake domain of Tat specifically inhibited E2-dependent reporter gene expression in COS-7 cells. Treatment of cervical intraepithelial neoplasia cells having episomally replicating HPV-31 DNA with this Tat-E2R protein led to a dose-dependent loss of HPV DNA copies and inhibition of cell growth. Tat-mediated delivery can be a valuable tool for assessing protein function and may allow the development of novel therapeutic proteins having intracellular targets.

The papillomavirus E2 protein is a DNA binding protein that regulates viral transcription and replication. E2 binds DNA as a dimer. Recent crystallographic data for E2 complexed to DNA revealed that novel peptide structures in E2 mediated dimerization and DNA binding. To identify important features of these motifs we have used limited proteolysis and urea denaturation as biochemical probes for structure, applying these techniques to E2 alone, E2 bound to DNA, cross-linked products, and mutants that were targeted at Trp360, a contact point along the dimer interface. DNA binding stabilized E2 structure, shifting the point at which it denatures from 5 to 7.6 M urea. In contrast, Trp360 mutant proteins, while dimeric, were more sensitive to denaturation by urea when bound to DNA. The most striking results came from uv cross-linking studies in which Trp360 was targeted as the site of cross-linking. Ultraviolet cross-linking dramatically increased the resistance of E2 to proteolysis regardless of the protease tested and with no deleterious effect on the affinity of E2 for DNA. Cross-linking through Cys356 with bismaleimidohexane did not promote stabilization. The ability to stabilize or destabilize E2 by Trp360-targeted modifications demonstrates the importance of the Trp360-Trp360 interaction, which may represent a general feature of the beta-barrel motif.

Mullerian inhibiting substance (MIS)/anti-Mullerian hormone is a differentiation factor that causes regression of the Mullerian duct in the developing male fetus and an apparent sex reversal of the fetal ovary when inappropriately exposed to it. The purified product is a 140-kilodalton glycoprotein composed of two identical subunits. We show that a C-terminal fragment of MIS, which shares homology with transforming growth factor-beta, causes regression of the Mullerian duct and inhibits the biosynthesis of aromatase in the fetal ovary. However, both activities are enhanced dramatically by addition of the N-terminal portion of MIS. Under conditions where potentiation occurs, the N- and C-terminal domains of MIS reassociate. These results indicate that the N-terminus of MIS, unlike that of the other members of the transforming growth factor-beta family, plays a role in maintaining the biological activity of the C-terminus.

The gene coding for human non-pancreatic phospholipase A2 (npPLA2) was cloned in a eukaryotic expression vector and transfected into chinese hamster ovary (CHO) cells. A number of cell lines stably expressing npPLA2 were obtained. Northern analysis of these cell lines showed an abundant transcript of expected size 1200 nt. The recombinant enzyme was efficiently secreted in quantities up to 400 micrograms npPLA2 per liter culture medium in the most productive cell lines. npPLA2 was purified to homogeneity from conditioned medium as previously described (1). The recombinant npPLA2 migrated by SDS--PAGE as a single band with an apparent mass of 14,000. The recombinant enzyme displayed the pH-optimum, calcium dependence and substrate preference that were characteristic of the human platelet and synovial fluid enzymes.

A 36 kDa calcium/phospholipid binding protein in human placenta was identified as VAC-beta (annexin-8) by a combination of immunological and peptide mapping analyses. The protein is a minor product in placenta, accounting for less than 1% of extracted annexins. From 150 g of tissue, only 100 micrograms of the protein was isolated. By anion-exchange chromatography on diethylaminoethyl-cellulose annexin-8 coeluted with annexin-3. By gel filtration, the protein chromatographed as a broad peak, where half the product eluted as a monomer and half eluted as a heterodimer that was associated with a 10 kDa subunit. The combination of annexin-8 being a minor component in standard annexin preparations and it co-eluting with annexin-3 by ion exchange chromatography are likely to account for the failure of other labs to characterize the product.

Anti-Müllerian hormone (AMH), also known as Müllerian-inhibiting substance or factor, has previously been shown to sex-reverse the steroidogenic pattern of fetal mammalian ovaries through repression of aromatase biosynthesis. Study of the ontogeny of the response of cyclic AMP-stimulated aromatase activity of rat fetal ovaries to AMH has allowed us to develop a quantitative bioassay for the hormone. Linear responses as a function of the logarithm of AMH concentration were observed over ranges of 0.2-7.5 micrograms/ml for the bovine protein and 0.15-2 micrograms/ml for the human protein, with a maximal decrease in aromatase activity of 90% for both proteins. Under the same in vitro conditions, AMH treatment did not affect cyclic AMP-stimulated fetal rat testicular aromatase activity. Partially purified chick AMH also decreased rat ovarian aromatase activity, allowing us to use this test to study AMH ontogeny in chick gonads. Analysis of the species specificity of AMH repression of ovarian aromatase activity indicated that turtle and rat fetal ovaries responded to AMH of other vertebrate classes, whereas aromatase activity of chick embryo ovaries could be repressed only by the homospecific hormone.

Recombinant soluble CD4 (rsCD4) has potent antiviral activity against cell line-adapted isolates of the human immunodeficiency virus type 1 (HIV-1) but low activity toward HIV-1 primary isolates from patients. A simple hypothesis proposed to explain this discrepancy, which questions the therapeutic utility of soluble CD4-based approaches, is that the major envelope glycoprotein, gp120, of patient virus has lower affinity for CD4 than does gp120 from laboratory viruses. To test this hypothesis, we have produced pairs of low- and high-passage HIV-1 isolates which, depending on culture passage history, display dramatically different sensitivities to neutralization by rsCD4. Here, we present evidence that the HIV-1 major envelope glycoprotein cDNAs cloned from one such isolate pair show only minor differences in their deduced gp120 primary structures, and these occur outside regions previously shown to be involved in CD4 interactions. In addition, recombinant gp120 from a low-passage rsCD4-resistant patient virus binds rsCD4 with high affinity, equal to that previously measured for recombinant gp120 from high-passage cell line-adapted virus isolates. These data indicate that differences in CD4-gp120 affinity do not account for rsCD4 resistance in HIV-1 recently isolated from patients.

The bovine papillomavirus E2 protein regulates viral transcription by binding as a dimer to the DNA sequence ACCGN4CGGT. The dimerization and DNA-binding properties are localized within its carboxy-terminal 85 amino acids (325-410). Utilizing random mutagenesis coupled with phenotypic selection in yeast, functionally important amino acids in the DNA-binding domain were identified. Four trans-activation defective point mutants within a short segment (amino acids 337-344) were DNA binding defective but dimeric. The mutation of a conserved tryptophan to serine also eliminated DNA binding, but loss of dimerization was implicated because addition of dimeric monoclonal antibody complemented this defect. A simple assay for E2 dimerization was developed using UV irradiation to produce an interchain cross-link within a dimer. No heterodimeric complexes were formed when pools of E2 of varying lengths were mixed, and only proteins with tryptophan at position 360 could be UV cross-linked. Peptide mapping of irradiated E2 protein localized the cross-link to an 18-amino-acid region bracketing this tryptophan. Substitutions for this tryptophan demonstrated the requirement for a hydrophobic residue at this position, but surprisingly, even alanine was functional. Replacement of this tryptophan with three polar amino acids or glycine eliminated DNA-binding activity, but addition of dimeric monoclonal antibody restored this function. The amino acids that were identified as being involved in DNA contact and dimerization imply that these functions are mediated by novel binding motifs.

Our previous studies have demonstrated that cultured human endothelial cells (EC) provide costimulation to PHA-activated CD4+ T cells, measured as augmentation of IL-2 synthesis, through a cell contact-department pathway. Here we show that fixed and living EC provide comparable degrees of costimulation to CD4+ T cell populations, indicating that EC costimulation does not depend upon active metabolism. EC achieve these effects in part by utilizing lymphocyte function-associated antigen-3 (LFA-3) to interact with T cell CD2 as shown by observations that EC augmentation of IL-2 is partially (50-70%) blocked by eight of eight mAb tested which recognize LFA-3; that purified phosphatidylinositol-linked LFA-3 (PI-LFA-3) can also provide costimulation to CD4+ T cells; and that there is a delay of the EC effect on CD4+ T cells which express low levels of CD2 compared to those which express high levels of CD2. However, three lines of evidence suggest that EC also utilize at least one additional ligand. First, there is incomplete replacement of the EC effect by PI-LFA-3 such that the costimulatory ability of EC combined with PI-LFA-3 is additive at all concentrations of PI-LFA-3 tested. Second, costimulation by PI-LFA-3, but not by EC, is fully inhibited by anti-CD2 or anti-LFA-3 mAb. Finally, costimulation by PI-LFA-3, but not by EC, is completely suppressed by cyclosporine A. We have not formally identified the second ligand but it does not appear to be intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD44, or B7/BB1.

We have used chemically cross-linked dimers, trimers, and tetramers of lymphocyte function-associated antigen-3 (LFA-3) to study the role of multivalency in the interaction of the protein with its receptor, CD2. The cross-linked adducts showed enhanced activity in systems where LFA-3 has been shown to (i) block LFA-3/CD2 interactions in a rosetting assay and (ii) provide through the CD2 on peripheral blood lymphocytes a trigger for T-cell proliferation. The level of increase was directly related to the valency state of the multimers. In the rosetting assay, the dimers, trimers, and tetramers, by weight, exhibited 15-, 150-, and 430-fold increases in activity over monomeric LFA-3. In the proliferation assay, the tetramer produced a 6-fold increase in thymidine incorporation at 0.06 micrograms/ml, the trimer was 100 times less active than the tetramer, and the dimer and monomer were inactive. The LFA-3 multimers were generated using a three-step cross-linking chemistry that was targeted at the carbohydrates on LFA-3. With this procedure over 60% of the starting protein was converted into multimers with no effect on function. The cross-linking approach should be applicable to other surface antigens, providing a simple method for analyzing multivalent interactions.

We have developed a three-step cross-linking procedure that is specifically targeted at the carbohydrate on a protein and applied it to CD4 as a model system for studying the role of multivalent interactions in function. In the first step CD4 was oxidized with periodate, creating aldehydes that served as targets for the subsequent chemistry. Next the aldehydes were modified with cystamine, converting the reactive group into a thiol. Finally cross-linking through the thiol moiety was generated with the homobifunctional cross-linker bismaleimidohexane. With this procedure, approximately 60% of the CD4 was converted into higher molecular weight complexes that were soluble and retained function as assessed by glycoprotein gp120 binding activity. CD4 dimers and tetramers by mass were 4 and 15 times as active as CD4 monomer in blocking virus infection with HTLV-IIIB in an in vitro cellular assay. The cross-linking chemistry provides an efficient method for producing homomultimers of a glycoprotein.

The solubility of guanidine hydrochloride in ethanol and conversely the low solubility of proteins have been used as the basis for a procedure for recovering proteins from guanidine-containing solutions by selective precipitation. Yields of greater than 94% were observed with as little as 28 ng protein and of 98% for larger quantities of protein up to 20 mg/ml. The precipitations were independent of molecular weight for proteins in the range of 6-100 kDa and could be run in as little as 5 min at room temperature. Unlike the conventional desalting methods for removing guanidine, ethanol precipitation is rapid, efficient, and can be applied simultaneously to a large number of samples. The approach should have a wide range of applications.

We evaluated the distribution of lipocortin-1 immunoreactivity in 118 immature or mature human hypophyses using the peroxidase-antiperoxidase (PAP) technique with a polyclonal rabbit antiserum against lipocortin-1. Serial sections were evaluated for five pituitary hormones and S-100 protein immunoreactivity to compare their distributions with that of lipocortin-1. Scattered or moderate numbers of cells exhibited lipocortin-1 immunoreactivity in the pars distalis of 89 subjects ranging in age from 27 weeks' gestation to 83 years. Seven immature and seven aged specimens exhibited no immunostaining, while 15 specimens from older individuals exhibited only rare immunostaining. Immunostaining did not appear to co-localize selectively with any specific pituitary hormone, although the distribution of immunoreactivity did overlap that of some corticotrophs and was seen in elongated processes of S-100-containing folliculostellate cells. Lipocortin-1 was also found in epithelial cells lining colloid cysts of the residual pars intermedia in 115 of 118 pituitaries ranging in age from 23 weeks' gestation to 83 years. In many intermediate lobe cysts, lipocortin-1 exhibited a pattern of immunoreactivity that partially overlapped the distribution of S-100 protein immunostaining, although the pattern was not identical. Pre-absorption of anti-lipocortin-1 antiserum with lipocortin-1-coupled Sepharose-4B immunoreactivity resulted in loss of immunoreactivity in both lobes. No lipocortin-1 immunoreactivity was seen in the neurohypophysis.