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Bone morphogenetic protein (BMP) signalling is key to many developmental processes, including early regionalisation of the ectoderm. The neural crest is induced here by a combination of BMP and Wnt signals from nearby tissues with many secreted factors contributing to its initial specification at the neural plate border. Gremlin 1 (Grem1) is a secreted BMP antagonist expressed in the neural crest in Xenopus laevis but its function here is unknown. As well as binding BMPs, Grem1 has been shown to interact with heparan sulfate proteoglycans (HSPGs), a family of cell surface macromolecules that regulate a diverse array of signalling molecules by affecting their availability and mode of action. This study describes the impact of HSPGs on the function of Grem1 in neural crest induction. It shows for the first time that Grem1 is required for neural crest development in a two-step process comprising an early HSPG-independent, followed by a late HSPG-dependent phase., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We have previously shown that the heterodimeric cytokine interleukin-12, and the homodimer of its larger subunit p40, both bind to heparin and heparan sulfate with relatively high affinity. In the present study we characterised these interactions using a series of chemically modified heparins as competitive inhibitors. Human interleukin-12 and p40 homodimer show indistinguishable binding profiles with a panel of heparin derivatives, but that of murine interleukin-12 is distinct. Heparin markedly protects the human and murine p40 subunits, but not the p35 subunits, from cleavage by the bacterial endoprotease LysC, further implicating the larger subunit as the location of the heparin binding site. Moreover the essential role of the carboxyterminal D3 domain in heparin binding is established by the failure of a truncated construct of the p40 subunit lacking this domain to bind. Predictive docking calculations indicate that a cluster of basic residues at the tip of the exposed C'D' loop within D3 is important in heparin binding. However since the human and murine C'D' loops differ considerably in length, the mode and three dimensional orientation of heparin binding are likely to differ substantially between the human and murine p40s. Thus overall the binding of IL-12 via its p40 subunit to heparin-related polysaccharides of the extracellular matrix appears to be functionally important since it has been conserved across mammalian species despite this structural divergence., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Of the circa 40 cytokines of the TGF-β superfamily, around a third are currently known to bind to heparin and heparan sulphate. This includes TGF-β1, TGF-β2, certain bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs), as well as GDNF and two of its close homologues. Experimental studies of their heparin/HS binding sites reveal a diversity of locations around the shared cystine-knot protein fold. The activities of the TGF-β cytokines in controlling proliferation, differentiation and survival in a range of cell types are in part regulated by a number of specific, secreted BMP antagonist proteins. These vary in structure but seven belong to the CAN or DAN family, which shares the TGF-β type cystine-knot domain. Other antagonists are more distant members of the TGF-β superfamily. It is emerging that the majority, but not all, of the antagonists are also heparin binding proteins. Any future exploitation of the TGF-β cytokines in the therapy of chronic diseases will need to fully consider their interactions with glycosaminoglycans and the implications of this in terms of their bioavailability and biological activity.

Gremlin-1, a bone morphogenetic protein (BMP) antagonist, has essential roles in kidney and limb bone development, and is important in chronic diseases including tissue fibrosis. It also functions as an activating ligand of the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2), and binds strongly to the sulfated polysaccharide, heparin. Here we investigated the extent to which gremlin binds to the related polysaccharide heparan sulfate (HS), which unlike heparin is widely distributed spread within tissues. We determined that both highly sulfated HS and kidney HS are able to partially compete for the binding of heparin to gremlin, whereas low sulfated HS is a poor competitor. In further investigations of the interaction between gremlin and HS, we found that wild-type gremlin is able to bind broadly across the various regions of kidney in an HS-dependent manner, with particularly intense binding to tubular structures in the renal cortex. In a model of chronic kidney disease, fibrotic changes in the kidney result in a loss of gremlin binding sites. Gremlin mutants with reduced affinity for heparin showed negligible binding under the same conditions. These mutants nonetheless remain functional as BMP antagonists on C2C12 myoblastic cells transfected with a Smad 1 reporter gene construct. Overall our findings indicate that on secretion, gremlin will bind to HS structures on the cell surface and in the extracellular matrix, thus providing for a localised reservoir which can modulate BMP activity in a temporospatially restricted manner. Although binding of heparin/HS to gremlin has been shown elsewhere to be necessary for gremlin activation of VEGFR2, this does not appear to be essential for BMP antagonism by gremlin. Thus these sulfated polysaccharides differentially regulate the activities of gremlin., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Gremlin is a member of the CAN (cerberus and DAN) family of secreted BMP (bone morphogenetic protein) antagonists and also an agonist of VEGF (vascular endothelial growth factor) receptor-2. It is critical in limb skeleton and kidney development and is re-expressed during tissue fibrosis. Gremlin binds strongly to heparin and heparan sulfate and, in the present study, we sought to investigate its heparin-binding site. In order to explore a putative non-contiguous binding site predicted by computational molecular modelling, we substituted a total of 11 key arginines and lysines located in three basic residue sequence clusters with homologous sequences from cerberus and DAN (differential screening selected gene abberative in neuroblastoma), CAN proteins which lack basic residues in these positions. A panel of six Myc-tagged gremlin mutants, MGR-1-MGR-6 (MGR, mutant gremlin), each containing different combinations of targeted substitutions, all showed markedly reduced affinity for heparin as demonstrated by their NaCl elution on heparin affinity chromatography, thus verifying our predictions. Both MGR-5 and MGR-6 retained BMP-4-binding activity comparable to that of wild-type gremlin. Low-molecular-mass heparin neither promoted nor inhibited BMP-4 binding. Finally, glutaraldehyde cross-linking demonstrated that gremlin forms non-covalent dimers, similar behaviour to that of DAN and also PRDC (protein related to cerberus and DAN), another CAN protein. The resulting dimer would possess two heparin-binding sites, each running along an exposed surface on the second β-strand finger loop of one of the monomers., (© 2015 Authors; published by Portland Press Limited.)

The bone morphogenetic proteins (BMPs) and the growth and differentiation factors comprise a single family of some 20 homologous, dimeric cytokines which share the cystine-knot domain typical of the TGF-β superfamily. They control the differentiation and activity of a range of cell types, including many outside bone and cartilage. They serve as developmental morphogens, but are also important in chronic pathologies, including tissue fibrosis and cancer. One mechanism for enabling tight spatiotemporal control of their activities is through a number of antagonist proteins, including Noggin, Follistatin, Chordin, Twisted gastrulation (TSG), and the seven members of the Cerberus and Dan family. These antagonists are secreted proteins that bind selectively to particular BMPs with high affinity, thereby blocking receptor engagement and signaling. Most of these antagonists also possess a TGF-β cystine-knot domain. Here, we discuss current knowledge and understanding of the structures and activities of the BMPs and their antagonists, with a particular focus on the latter proteins. Recent advances in structural biology of BMP antagonists have begun the process of elucidating the molecular basis of their activity, displaying a surprising variety between the modes of action of these closely related proteins. We also discuss the interactions of the antagonists with the glycosaminoglycan heparan sulfate, which is found ubiquitously on cell surfaces and in the extracellular matrix., (© 2015 Elsevier Inc. All rights reserved.)

Mucosal boosting of BCG-immunised individuals with a subunit tuberculosis (TB) vaccine would be highly desirable, considering that the lungs are the principal port of entry for Mycobacterium tuberculosis (MTB) and the site of the primary infection and reactivation. However, the main roadblock for subunit TB vaccine development is the lack of suitable adjuvants that could induce robust local and systemic immune responses. Here, we describe a novel vaccine delivery system that was designed to mimic, in part, the MTB pathogen itself. The surface of yellow carnauba wax nanoparticles was coated with the highly immunogenic Ag85B Ag of MTB and they were directed to the alveolar epithelial surfaces by the incorporation of the heparin-binding hemagglutinin adhesion (HBHA) protein. Our results showed that the i.n. immunisation of BCG-primed BALB/c mice with nanoparticles adsorbed with Ag85B-HBHA (Nano-AH vaccine) induced robust humoral and cellular immune responses and IFN-γ production, and multifunctional CD4⁺ T cells expressing IFN-γ, IL-2 and TNF-α. Mice challenged with H37Rv MTB had a significantly reduced bacterial load in their lungs when compared with controls immunised with BCG alone. We therefore conclude that this immunisation approach is an effective means of boosting the BCG-induced anti-TB immunity.

The q arm of chromosome 1 is frequently amplified at the gene level in breast cancer. Since the significance of this is unclear we investigated whether 1q genes are overexpressed in this disease. The cDNA levels of 1q-located genes were analysed in a search for overexpressed genes. 26 genes mapping to the 1q arm show highly significant (P≤0.01) overexpression of transcripts in breast cancer compared to normal breast tissue. Amongst those showing the highest levels of overexpression in both expressed sequence tag (EST) and serial analysis of gene expression (SAGE) databases was enzyme quiescin Q6 sulfhydryl oxidase 1 (QSOX1). We investigated QSOX1 cDNA derived from T47D breast carcinoma cells by RT-PCR and 3'-RACE PCR and identified a novel extended form of QSOX1 transcript, containing a long 3'UTR, nearly double the size of the previously reported QSOX1 cDNA, and confirmed its 3' end nucleotide sequence using RACE-PCR. We also used quantitative real-time PCR to analyse a panel of cDNAs derived from 50 clinically-graded normal and malignant breast tissue samples for the expression of QSOX1 mRNAs. QSOX1 transcription was elevated in an increasing proportion in the grade 2 and grade 3 tumours (graded according to the Nottingham prognostic index), with 10 of the 15 grade 3 tumours (67%) examined exceeding the normal range. There was a significant correlation between relative transcript level and clinical grade (P≤0.01) for all qPCR primer sets tested. QSOX1 mRNA levels, based on SAGE expression data, did not correlate with either Estrogen Receptor (ER) or Epidermal Growth Factor Receptor 2 (ErbB-2 or HER2/neu) expression. Our data indicate that QSOX1 is a potential new prognostic marker which may prove of use in the staging of breast tumours and the stratification of breast cancer patients.

The BMPs (bone morphogenetic proteins) and the GDFs (growth and differentiation factors) together form a single family of cystine-knot cytokines, sharing the characteristic fold of the TGFbeta (transforming growth factor-beta) superfamily. Besides the ability to induce bone formation, which gave the BMPs their name, the BMP/GDFs display morphogenetic activities in the development of a wide range of tissues. BMP/GDF homo- and hetero-dimers interact with combinations of type I and type II receptor dimers to produce multiple possible signalling complexes, leading to the activation of one of two competing sets of SMAD transcription factors. BMP/GDFs have highly specific and localized functions. These are regulated in a number of ways, including the developmental restriction of BMP/GDF expression and through the secretion of several specific BMP antagonist proteins that bind with high affinity to the cytokines. Curiously, a number of these antagonists are also members of the TGF-beta superfamily. Finally a number of both the BMP/GDFs and their antagonists interact with the heparan sulphate side chains of cell-surface and extracellular-matrix proteoglycans.

Recombinant human betacellulin binds strongly to heparin, requiring of the order of 0.8 M NaCl for its elution from a heparin affinity matrix. This is in complete contrast to the prototypic member of its cytokine superfamily, epidermal growth factor, which fails to bind to the column at physiological pH and strength. We used a well-established heparin binding ELISA to demonstrate that fucoidan and a highly sulfated variant of heparan sulfate compete strongly for heparin binding. Low sulfated heparan sulfates and also chondroitin sulfates are weaker competitors. Moreover, although competitive activity is reduced by selective desulfation, residual binding to extensively desulfated heparin remains. Even carboxyl reduction followed by extensive desulfation does not completely remove activity. We further demonstrate that both hyaluronic acid and the E. coli capsular polysaccharide K5, both of which are unsulfated polysaccharides with unbranched chains of alternating N-acetylglucosamine linked beta(1-4) to glucuronic acid, are also capable of a limited degree of competition with heparin. Heparin protects betacellulin from proteolysis by LysC, but K5 polysaccharide does not. Betacellulin possesses a prominent cluster of basic residues, which is likely to constitute a binding site for sulfated polysaccharides, but the binding of nonsulfated polysaccharides may take place at a different site.

GDNF (glial cell-line-derived neurotrophic factor), and the closely related cytokines artemin and neurturin, bind strongly to heparin. Deletion of a basic amino-acid-rich sequence of 16 residues N-terminal to the first cysteine of the transforming growth factor beta domain of GDNF results in a marked reduction in heparin binding, whereas removal of a neighbouring sequence, and replacement of pairs of other basic residues with alanine had no effect. The heparin-binding sequence is quite distinct from the binding site for the high affinity GDNF polypeptide receptor, GFRalpha1 (GDNF family receptor alpha1), and heparin-bound GDNF is able to bind GFRalpha1 simultaneously. The heparin-binding sequence of GDNF is dispensable both for GFRalpha1 binding, and for activity for in vitro neurite outgrowth assay. Surprisingly, the observed inhibition of GDNF bioactivity with the wild-type protein in this assay was still found with the deletion mutant lacking the heparin-binding sequence. Heparin neither inhibits nor potentiates GDNF-GFRalpha1 interaction, and the extracellular domain of GFRalpha1 does not bind to heparin itself, precluding heparin cross-bridging of cytokine and receptor polypeptides. The role of heparin and heparan sulfate in GDNF signalling remains unclear, but the present study indicates that it does not occur in the first step of the pathway, namely GDNF-GFRalpha1 engagement.

The TGF-beta (transforming growth factor-beta) cytokine superfamily in mammals contains some 30 members. These dimeric proteins are characterized by a strongly conserved cystine knot-based structure. They regulate the proliferation, differentiation and migration of many cell types, and therefore have important roles in morphogenesis, organogenesis, tissue maintenance and wound healing. Thus far, around one-quarter of these cytokines have been shown to bind to heparin and heparan sulphate. Well-established examples are the TGF-beta isoforms 1 and 2, and the BMPs (bone morphogenetic proteins) -2 and -4. In studies in my laboratory, we have shown that GDNF (glial-cell-line-derived neurotrophic factor) and its close relatives neurturin and artemin bind to heparin and heparan sulphate with high affinity. We have reported previously that binding of GDNF is highly dependent on the presence of 2-O-sulphate groups. More recently, we and others have been investigating the heparin/heparan sulphate-binding properties of BMP-7, which is a representative of a distinct BMP subgroup from that of BMPs -2 and -4. Interestingly, several of the various specific BMP antagonist proteins also bind to heparin and heparan sulphate. Much remains to be learnt about the nature and role of glycosaminoglycan interactions in the TGF-beta superfamily, but current work suggests that these cytokines do not share a single highly conserved heparin/heparan sulphate-binding site.

The defining characteristic of the glycoproteins known as proteoglycans is the presence of O-linked acidic polysaccharides known as GAGs (glycosaminoglycans). The backbone of these linear polysaccharides is a repeating disaccharide, comprising N-acetyl hexosamine alternating with beta-D-glucuronic acid, alpha-L-iduronic acid, or galactose. For some GAGs, partial deacetylation, epimerization of glucuronic acid, and substitution with N- and O-sulphates result in highly complex, heterogeneous structures. The interactions with proteins through which GAGs exert their biological effects depend on the resulting sequences. Some proteins, for example antithrombin, have highly specific sequence requirements for their GAG ligand [in this case heparin or HS (heparan sulphate)]; others, for example the fibroblast growth factors, are less demanding. GAGs, in particular HS, play a role as co-receptors for some cytokines. In addition, HS is thought to be important for the localization of cytokines, acting both as a tissue store and as a mediator of morphogen gradient formation in development. The structural determinants of GAG-cytokine interactions are therefore clearly important to understanding the biology of development, wound healing and the immune system. No single paradigm has been identified for such interactions, and the search for general principles underlying involvement of GAGs in cytokine function is at an early stage.

We report ELISA studies of the glycosaminoglycan binding properties of recombinant human glial cell line-derived neurotrophic factor (GDNF). We demonstrate relatively high affinity binding as soluble heparin competes with an IC50 of 0.1 micro g/ml. The binding of GDNF to heparin is particularly dependent on the presence of 2-O-sulfate groups. Highly sulfated heparan sulfate is also an effective competitor for GDNF binding. We also show that heparin at low concentrations protects GDNF from proteolytic modification by an endoprotease and also promotes the binding of GDNF to its receptor polypeptide, GFRalpha1. In both of these actions, 2-O-desulfated heparin is less effective. Considered overall, these findings provide strong support for a hypothesis that the bioactivity of GDNF during prenatal development is essentially dependent on the binding of this growth factor to 2-O-sulfate-rich heparin-related glycosaminoglycan.

Glial cell line-derived growth factor (GDNF) is a cytokine of the transforming growth factor (TGF)-beta family with potent neuroprotective activity. Clinical trials of recombinant GDNF in advanced Parkinson's disease are currently under way. It is known that mice homozygous for disruption of the gene encoding heparan sulphate 2-O-sulphotransferase die perinatally, due to the complete absence of kidneys. Similar phenotypes arise from targeted disruption of the genes encoding either GDNF, or its receptors, GFRalpha1 and c-Ret. It may therefore be proposed that GDNF normally binds to 2-O-sulphate-rich heparan sulphate within kidney progenitor tissues, and that this interaction is essential for its activity in kidney development. In support of this hypothesis we have shown in ELISA studies that GNDF binds to heparin and heparan sulphate. This binding is unusually sensitive to the chemical 2-O-desulphation, and promotes the binding of GNDF to GFRalpha1.

We show, using a murine NK cell line which responds quantitatively to rmIL-12, that treatment with ChABCase, but not other GAGases, results in substantial reductions in the secretion of gamma-IFN. Likewise, treatment of the cells with a beta-D-xyloside inhibitor of proteoglycan biosynthesis inhibits this cytokine response. In both treatments, the addition of soluble, exogenous GAGs does not relieve the inhibition of gamma-IFN secretion. We also demonstrate by ELISA that rmIL-12 binds to CS B. Overall, our studies on this in vitro cellular model of the initiation of Th1 immune responses indicate a major role for cell-surface, iduronate-rich, CS proteoglycan in the biological activity of IL-12.

We establish, using an ELISA approach, that recombinant human and murine IL-6 bind to an immobilized heparin-BSA complex. In the case of human IL-6, this binding is displaceable by soluble heparin, IC(50) approximately 2 microg/ml, corresponding to approximately 200 nM. This binding is specific because chondroitin sulfates B and C fail to compete, whereas chondroitin sulfate A and several heparan sulfates are weak inhibitors. Of a range of chemically modified heparins examined, the strongest competitor was the 2-O:-desulfated product, but even this showed a considerably reduced IC(50) ( approximately 30 microg/ml). The epitopes of five IL-6-specific mAbs were still accessible in heparin-bound IL-6, and the dimer formed from the association of rIL-6 with its truncated soluble receptor polypeptide, srIL-6alpha, still bound to heparin. Further analysis showed that heparin competed partially and weakly with the binding of srIL-6 to IL-6; however, it competed strongly for the binding of the rIL-6/srIL-6Ralpha dimer, to soluble glycoprotein 130. In studies of the proliferation of IL-6-sensitive Ba/F3 cells expressing glycoprotein 130, we were unable to detect any effect of either the removal of cell surface heparan sulfate, or addition of soluble heparin. By contrast, heparin was able to protect IL-6 from digestion by the bacterial endoproteinase Lys-C. Overall, our findings show that IL-6 is a heparin-binding cytokine. This interaction will tend to retain IL-6 close to its sites of secretion in the tissues by binding to heparin-like glycosaminoglycans, thus favoring a paracrine mode of activity. Moreover, this binding may serve to protect the IL-6 from proteolytic degradation.

Protein tyrosine phosphatases play a critical role in controlling tyrosine phosphorylation levels of proteins. Ischemia induces changes in tyrosine phosphorylation. As part of our investigations of the mechanisms responsible for these changes, we studied the effects of cerebral hypoxia-ischemia in 7-day-old (P7) and P21 rat brains on expression of the STEP (striatal enriched phosphatase) family of protein tyrosine phosphatases. P7 and P21 rats were subjected to unilateral hypoxia-ischemia, and brains were analyzed at various intervals of recovery for the presence of STEP. Hypoxia-ischemia induced the formation of a low Mr isoform of STEP, STEP33, in the ipsilateral (damaged) hemisphere but not in the contralateral (undamaged) side. STEP33 produced as a result of ischemia was located exclusively in the cell soluble fraction. In P21 rats, the ischemia-induced elevation in STEP33 was delayed relative to P7 rats. STEP33 was produced by digestion of postsynaptic densities with calpain I and by exposure of NT2/D1 cells expressing STEP to the calcium ionophore A23187. The results suggest that ischemia-induced calcium influx results in the calcium-dependent proteolysis of membrane-associated STEP61 and the concomitant release of STEP33 into the cytoplasm.

Many viruses have evolved to exploit cell-surface glycosaminoglycans (GAG), particularly heparan sulfate, to facilitate their attachment and infection of host cells. Here, the case for the involvement of heparan sulfate GAG in cellular infection by human immunodeficiency virus Type 1 (HIV-1) compared with herpes simplex virus Type 1 (HSV-1) is re-examined. It is shown that HIV-1 infection is facilitated by heparan sulfate GAG in only one of three highly permissive cell lines tested, whereas HSV-1 infection is facilitated to varying extents in all three. To evaluate the physiological relevance of these findings, primary peripheral blood lymphocytes (PBL), the physiological host for HIV-1, were examined. It was found that treatment of PBL with heparitinase, to remove any traces of heparan sulfate GAG, did not alter their sensitivity to infection by either lymphocyte-tropic, X4-type strain HIV-1IIIB, nor the monocyte-tropic, R5-type strain, HIV-1Ba-L. It is concluded that heparan sulfate GAG has little physiological role in the infection of lymphocytes by HIV-1 and that evidence derived from studies on immortalized cell lines suggesting a significant role must be interpreted with caution.

Using an ELISA approach, we demonstrate that recombinant human IL-12 (rhIL-12) binds strongly to an immobilized heparin-BSA complex. This binding is completely displaceable with soluble heparin, IC50 approximately 0.1 microg/ml, corresponding to approximately 10 nM. By interpolation with our previous findings, this indicates an affinity for heparin greater than that of antithrombin III and comparable with that of FGF-2, two high-affinity heparin-binding proteins. Recombinant murine IL-12 also binds strongly to heparin. The binding of rhIL-12 to heparin shows specificity because chondroitin sulfates A and C fail to compete, whereas chondroitin B inhibits weakly. A highly sulfated heparan sulfate is a strong competitor, whereas other heparan sulfates show weak or no activity. Small heparin fragments inhibit binding, although activity decreases with size. An octasaccharide pool derived by cleavage of heparin with nitrous acid is a significantly stronger inhibitor than its heparinase I-derived counterpart, further indicating structural specificity in the interaction between rhIL-12 and heparin. The binding of recombinant p40 to heparin appears indistinguishable from that of the IL-12 heterodimer, implying that the heparin binding site is largely if not solely located in this subunit. These results show for the first time that IL-12 is a heparin-binding cytokine, a property common to the other Th1-response-inducing cytokines, IFN-gamma and IL-2. Our findings strongly suggest that IL-12 will tend to be retained close to its sites of secretion in the tissues by binding to heparin-like glycosaminoglycans, thus favoring a paracrine role for IL-12.

We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, highly sulfated heparan sulfate and fucoidan. Here we show that IL-2 binding is dependent on heparin chain length, but with fragments as small as 15-mers retaining binding activity. The addition of exogenous heparin has no effect on the in vitro biological activity of IL-2. In addition soluble IL-2 receptor alpha and beta polypeptides do not compete with heparin for the binding of IL-2. IL-2 bound by heparin is still recognized by two IL-2 specific monoclonal antibodies, 3H9 and H2-8, whose epitopes lie in the amino terminal region. Murine IL-2 unlike its human counterpart fails to bind to heparin. Human IL-2 analogs with single amino acid substitutions at positions Lys43, Thr51, and Gln126 analogs no longer bind to heparin. By contrast the Arg38Ala analog retains heparin full heparin binding activity. These experimental findings together with molecular modeling studies suggest two putative heparin binding sites on human IL-2, one involving four basic residues, Lys48, Lys49, Lys54, and His55, and the other being a discontinuous site comprising Lys43, Lys64, Arg81, and Arg83. Neither of these two clusters is completely conserved in murine IL-2. Overall our data suggest that the binding of human IL-2 to heparin and heparan sulfate does not interfere with IL-2/IL-2 receptor interactions. Therefore, binding to glycosaminoglycan may be a mechanism for retaining the cytokine in an active form close to its site of secretion in the tissue, thus favoring a paracrine role for IL-2.

Postnatal rats at 7 and 21 days of age were subjected to unilateral hypoxia-ischemia (H/I) by right carotid artery ligation followed by 1.5 to 2 hours of hypoxia (8% oxygen). Brains were frozen at specific intervals of recovery from 0 to 24 hours. Western blots of samples of right and left forebrain were immunodeveloped with a monoclonal antibody specific for ubiquitin, RHUb1. An elevation of ubiquitin conjugate levels in the right compared with the left forebrain of 7-day-old animals was detectable immediately following H/I and increased by close to 60% of control level within 1 hour of recovery. The conjugate immunoreactivity remained at this level for 6 hours but had declined to control levels by 24 hours of recovery. No such increase was observed in response to hypoxia alone. Similar changes were observed in samples from the 21-day-old rat brain. However, the elevation of ubiquitin conjugate levels was of slower onset and persisted longer than observed for the 7-day-old animals. Immunocytochemical studies of brain fixed by immersion in formaldehyde/acetone/methanol showed that ubiquitin-like immunoreactivity was increased in the right, but not left, cerebral cortex and hippocampus of animals subjected to H/I. The data suggest that elevated ubiquitination may represent a neuroprotective response to H/I.

We have employed a direct radiolabel binding assay to investigate the interaction between3H-heparin and recombinant envelope glycoproteins, rgp120s, derived from several different isolates of HIV-1. Comparable dose-dependent binding is exhibited by rgp120s from isolates IIIB, GB8, MN and SF-2. Under identical experimental conditions the binding of3H-heparin to a recombinant soluble form of the cellular receptor for gp120, CD4, is negligible. The binding of3H-heparin to rgp120 is competed for by excess unlabeled heparin and certain other, but not all, glycosaminoglycan and chemically modified heparins. Of a range of such polysaccharides tested, ability to compete with3H-heparin for binding was strictly correlated with inhibition of HIV-1 replication in vitro. Those possessing potent anti-HIV-1 activity were effective competitors, whereas those having no or little anti-HIV-1 activity were poor competitors. Scatchard analysis indicates that the K d of the interaction between heparin and rgp120 is 10 nM. Binding studies conducted in increasing salt concentrations confirm that the interaction is ionic in nature. Synthetic 33-35 amino acid peptides based on the sequence of the V3 loop of gp120 also bind to heparin with high affinity. V3 loop peptides that are cyclized due to terminal cysteine residues show more selective binding than their uncyclized counterparts. Overall, these data demonstrate further that heparin exerts its anti-HIV-1 activity by binding to the envelope glycoprotein of HIV-1, rather than its cellular receptor, CD4. This study confirms that the V3 loop of gp120 is the site at which heparin exerts its anti-HIV-1 activity. Moreover, it reveals that high affinity binding to heparin is shared by all four rgp120s examined, despite amino acid substitutions within the V3 loop.

Polyamines have been implicated in a wide variety of functions including nucleic acid synthesis and protein synthesis. Their levels have been shown to increase in response to cell growth and differentiation. Use of polyamines as prognostic indicators of proliferative disease conditions has been hindered by the lack of suitable rapid and sensitive assays. We report the characterization of an anti-spermidine antibody, JSJ-1, with novel putrescine cross reactivity. JSJ-1 cross-reacts more strongly with putrescine (11%) than with spermine (6%). This suggests that the aminobutyl group common to both putrescine and spermidine is an important element in the antibody-antigen interaction. We have demonstrated that antibody-spermidine binding is effected by increased ionic strength. This finding is consistent with the antibody-antigen interaction being ionic. The JSJ-1 antibody has been successfully used to detect increased polyamine levels in clinical serum samples and identify those with increased polyamine levels.

We have developed an enzyme-linked immunosorbent assay (ELISA) approach for the study of interactions between cytokines and glycosaminoglycans. This involves, as solid phase, a synthetic heparin-bovine serum albumin (BSA) complex in which the heparin is coupled via its reducing terminus to the protein using sodium cyanoborohydride. We have investigated the sensitivity and specificity of this experimental technique, employing antithrombin (AT III) and fibroblast growth factor 2 (FGF-2) as well-characterized heparin binding proteins. Using this ELISA method, we have established that human recombinant interleukin (IL-2) binds to heparin in a concentration-dependent manner. Soluble heparin competes for the binding of IL-2 to the complex with 50% inhibition at 5 microg/ml. This IC50 value provides an estimate of the binding constant of around 0.5 microM. This value is at least two orders of magnitude larger than that for the binding of IL-2 to its dimeric and trimeric cell surface receptors, but similar to that for binding to the IL-2 receptor beta polypeptide acting alone. Our ELISA shows that in addition to soluble heparin, fuciodan also competes for IL-2 binding, but chondroitin sulfate and dermatan sulfate are inactive. Of six heparan sulfates tested, only one highly sulfated preparation competed for IL-2. The interaction between IL-2 and heparin-like glycosaminoglycans is likely to be an important mechanism for retaining IL-2 close to its sites of secretion, thus giving rise to localized concentration gradients in the tissues., (Copyright 1997 Academic Press Limited.)

The role of ubiquitin in development of the mammalian brain has been studied using a monoclonal antibody, RHUb1, specific for ubiquitin. Immunodevelopment of western blots of homogenate samples of the cerebral cortex, hippocampus and cerebellum prepared from animals of known postnatal age show marked developmental changes in conjugate level. Striking decreases in the level of a prominent conjugate of molecular weight 22,000, which is identified as ubiquitinated histone, are observed during the first postnatal week in the cerebral cortex and hippocampus, but not the cerebellum. A marked overall developmental decrease in the level of high-molecular-weight (> 40,000) ubiquitin conjugates which occurs predominantly during the third, but also the fourth, postnatal week is observed in all three regions. Immunocytochemical data obtained with the RHUb1 antibody show intense staining of neuronal perikarya, nuclei and dendrites in early postnatal cerebral cortex and hippocampus. Staining of pyramidal cell perikarya and dendrites is particularly prominent. The intensity of dendritic staining, particularly for the cerebral cortex, shows a striking decrease after postnatal day 14 and only faint dendritic staining is observed in the adult. In early postnatal cerebellum, immunoreactivity is predominantly nuclear, though some staining of the proximal regions of Purkinje cell dendrites is observed between postnatal days 4 and 19. As with the cerebral cortex and hippocampus, most of the ubiquitin reactivity is lost in adult animals. The loss of dendritic staining, particularly in the cerebral cortex, correlates with the decrease in the level of high-molecular-weight ubiquitin conjugates observed on the western blots. Immunodevelopment of western blots of a range of subcellular fractions prepared from developing rat forebrain shows that the developmental decrease in the level of high-molecular-weight ubiquitin conjugates is not uniform for all fractions. The decrease in conjugate level is most marked for the cell-soluble, mitochondrial and detergent-insoluble cytoskeletal fractions. Taken overall, the data suggest a role for ubiquitin in dendrite outgrowth and arborization, loss of dendritic ubiquitin immunoreactivity correlating with completion of these processes.

Heparin is one of several sulphated polysaccharides which potently inhibit replication of the human immunodeficiency virus type 1 (HIV-1) in cultures of CD4+ve human cells. The EC50 value is around 5 microg ml(-1). We have demonstrated that heparin binds to recombinant gp120, the envelope glycoprotein of HIV-1, at a site termed the V3 loop, or principle neutralizing domain, which consists of a disulphide-bridged loop of 32-35 amino acids particularly enriched with basic residues. Using a series of chemically modified heparins we have shown that there is structural specificity in the anti-HIV activity of heparin. Heparin is routinely used clinically as an anticoagulant, and has proved essentially non-toxic and well tolerated. Low anticoagulant derivatives of heparin which retain high anti-HIV-1 activities in vitro may be generated by several routes. Such preparations are ideal candidates for clinical investigation as potential novel therapeutic agents for use in combination with other drugs in the management of AIDS and HIV infection.

To enable the immunoassay of spermidine in tissue extracts, a monoclonal antibody, JAC-1, specific for free spermidine was raised, using a spermidine-ovalbumin conjugate as immunogen. This antibody was characterized, and found to possess a high degree of specificity for spermidine, with only 4% molar cross-reactivity with spermine. A competitive ELISA using this antibody was developed. This assay is able to detect as little as 10 pmol spermidine extracted from small, circa 10 mg, tissue samples. The assay is unaffected by the presence of up to a 4-fold molar ratio of spermine, whereas the corresponding spermine competitive ELISA is adversely affected by a 0.5-fold molar ratio of spermidine. The spermidine competitive ELISA using JAC-1 was used to estimate the spermidine content of several plant and animal tissue extracts and the results compared with HPLC data. This novel assay is a useful development in the assay of polyamines since, compared to routinely employed HPLC methods, it offers increased convenience, rapidity, and capacity for large numbers of samples.

Heparin is a potent inhibitor of HIV-1 replication, in addition to being a well-established inhibitor of blood coagulation. The major anticoagulant activity of heparin results from binding to the plasma protein antithrombin (AT). The high-affinity binding site for AT is a specific pentasaccharide sequence that is of low abundance and completely absent from the majority of heparin chains. We have examined the anti-HIV-1 activity of both conventional and low molecular weight heparins fractionated according to affinity for AT. The high- and low-affinity fractions, despite differing markedly in anticoagulant activity, are identical in their ability to bind to the envelope glycoprotein of HIV-1, and in their inhibitory effect on HIV-1 replication in vitro (EC50 1 and 8 micrograms/ml for conventional and low molecular weight fractions, respectively). Our study shows that the anti-HIV activity of heparin is independent of its antithrombin-mediated inhibition of coagulation proteases. Therefore, heparin preparations retaining full anti-HIV-1 activity in vitro but with greatly reduced anticoagulant activity may be readily produced for further clinical investigation in the prophylaxis and therapy of HIV infection.

Chemically modified heparins were tested for their activities in (i) inhibiting HIV-1 replication in vitro and (ii) inhibiting the binding to recombinant HIV-1 gp120 of monoclonal antibodies specific for the V3 loop. The results reveal that N-desulfation reduces activity, although this is largely restored on N-acetylation. Selective O-desulfation also markedly reduces activity, whereas carboxyl reduction has little effect. Overall these results show that the anti-HIV-1 activity of heparin does not depend simply on negative density, and indicate instead that particular structures, notably O-sulfates, are involved. Our studies reveal that for chemically modified heparins and heparin-derived fragments there is a striking correlation between anti-HIV-1 activity in vitro and binding to the V3 loop of gp120 in solid phase ELISA. This strongly suggests that the heparin exerts its anti-HIV-1 activity by binding to the V3 loop of gp120.

The pattern of ubiquitin-protein conjugates present in a range of adult rat forebrain subcellular fractions has been investigated by immunoblotting with a monoclonal antibody specific for ubiquitin and its conjugates. Each fraction contains a complex and characteristic pattern of ubiquitin conjugates. Many integral synaptic membrane proteins are ubiquitinated, including a subset of high M(r) (> 120 kD) concanavalin A-binding glycoproteins. Postsynaptic densities are also enriched in ubiquitin conjugates, the profile being distinct from that of synaptic membranes. These results suggest that many plasma membrane and synaptic proteins are ubiquitinated.

Objective: To investigate the binding of the sulphated polysaccharides, dextran sulphate and heparin, to CD4 and gp120 in order to examine the anti-HIV mechanisms of these compounds., Design: In order to study the molecular mechanisms involved, the binding of sulphated polysaccharides to recombinant (r) sCD4 and gp120 was investigated in solid-phase binding studies that employed various monoclonal antibodies directed against known epitopes on these proteins, including the V3 loop of gp120., Methods: The ability of sulphated polysaccharides to inhibit both the binding of gp120 to CD4 and the binding of the monoclonal antibodies was investigated by enzyme-linked immunosorbent assays., Results: It was demonstrated that dextran sulphate inhibits gp120-sCD4 binding at concentrations of 100 micrograms/ml, whereas heparin has no effect. Heparin does, however, block the binding to rgp120 of monoclonal antibodies recognizing epitopes in the V3 loop. Clinical low molecular weight heparin preparations are as active as unfractionated heparin in this regard. Pre-incubation of gp120 with excess sCD4 increases the potency of heparin in blocking the binding of V3 loop monoclonals severalfold., Conclusions: The modes of action of heparin and dextran sulphate differ. Dextran sulphate both inhibits CD4-gp120 binding and binds to the V3 loop of gp120. However, heparin is more selective and appears to function only by interfering with events involving the V3 loop that occur prior to HIV fusion with the plasma membrane.

Spermine-specific monoclonal antibodies were prepared by immunising mice with protein-spermine conjugates. A resulting monoclonal antibody, IAG-1, exhibited both high affinity for spermine (binding constant 5.5 x 10(7) M-1) and high specificity, cross-reacting only weakly with spermidine. Using this antibody a competitive ELISA was developed with a detection limit of 1 pmol. The assay has been used to quantify spermine content of plant tissues, without derivatization, and producing within 6 h of collection, values for the spermine content which are similar to published data obtained by HPLC.

The binding of interleukin (IL)-1 alpha, IL-1 beta, IL-2 and IL-6 to acidic polysaccharides was investigated by affinity chromatography of the recombinant, radioiodinated interleukins on columns of immobilized polysaccharide. Each interleukin showed selective binding retention. Overall heparin bound all four interleukins significantly, whereas chondroitin sulfate provided little retention. IL-1 alpha and IL-1 beta showed differential binding, with only the latter binding to hyaluronic acid. IL-2 was virtually completely retained on fucoidan. Noniodinated recombinant IL-2 bound similarly to fucoidan, and fucoidan was found to sequester IL-2 activity in a bioassay employing IL-2-dependent CTLL cells. In all other cases tested, interleukin retention was partial, implying that interleukin binding sites are sparsely distributed along the polysaccharide chains. These findings suggest that during the immune response, interleukins will tend to be retained at sites of secretion by interaction with glycosaminoglycans in the extracellular matrix and on cell surfaces.

Metabolic labeling of the murine T lymphoma cell line RDM-4 with [35S] sulfate results in intense incorporation into a cell-retained component of apparent Mr approximately 100,000. This macromolecule is identified as a glycoprotein by lectin chromatography. The sulfate is not incorporated as tyrosine sulfate. Release of the radiolabel by alkaline beta-elimination but not by endoglycosidase F is consistent with the sulfation of O- rather than N-linked oligosaccharides. The sulfated glycoprotein displays anomalous migration on SDS-PAGE in two respects: 1) the apparent Mr shifts from 115,000 to 87,000 on increasing the acrylamide concentration from 7 to 12%, and 2) on neuraminidase digestion migration is substantially reduced (apparent Mr 140,000). These properties indicate that the sulfated protein is both heavily glycosylated and extensively sialylated, and are characteristic of the lymphoid mucin, leukosialin (sialophorin, CD43). Specific labeling of the sialoglycoproteins of RDM-4 cells indicates that leukosialin, the most intensely labeled protein, comigrates with the sulfated protein on SDS-PAGE at varying acrylamide concentrations. Our data are therefore consistent with sulfation of at least some of the numerous O-linked oligosaccharides of this abundant glycoprotein in RDM-4 cells. No sulfation of CD43 in resting splenic T cells is observed.

Normal murine splenic T lymphocytes and T-lymphoma cells were incubated with [35S]sulphate in low-sulphate medium for 4 hr. Gel filtration and SDS-PAGE revealed that the radiolabelled macromolecules secreted by these cells were almost exclusively chondroitin sulphate and heparan sulphate proteoglycans of relatively low molecular weight (MW), 100,000-200,000. Triton X-100 extracts of the cells contained similar proteoglycans. Under the conditions employed the incorporation of radiolabel by cells grown in vivo was equally distributed between cell-retained and secreted fractions, whereas cells grown in vitro retained some 75% of incorporated label. In general heparan sulphate predominated over chondroitin sulphate in both secreted and cell-retained fractions. Cell extracts also contained a minor proportion of free glycosaminoglycan, which is almost exclusively heparan sulphate. These chains, like those incorporated into the proteoglycan, were around 12,000 MW. The T-lymphoma cells RDM-4, whether grown in vitro or in vivo, also incorporated a substantial proportion of [35S]sulphate into a single, cell-retained protein, 100,000 MW. No such radiolabelled protein was detectable in T cells.

The nature of the stromal cells in formalin-fixed paraffin-embedded material from 23 cerebellar haemangioblastomas was investigated using antisera to intermediate filaments (glial fibrillary acidic protein, vimentin and desmin), histiocytic markers (alpha 1-antitrypsin, alpha 1-antichymotrypsin and lysozyme), glycolytic enzymes (alpha and gamma enolase and aldolase C4) and the endothelial markers, factor VIII related antigen and Ulex europaeus I lectin. Most stromal cells stained positively for vimentin and the glycolytic enzymes. Occasional process-bearing cells within the stroma stained strongly for glial fibrillary acidic protein, alpha 1-antitrypsin and alpha 1-antichymotrypsin. No stromal cell staining for desmin, lysozyme or the endothelial markers was observed, although the latter stained the vascular endothelium within all neoplasms. The findings do not support previous suggestions of an endothelial or histiocytic origin for the stromal cells. They appear to be a heterogeneous population including entrapped reactive astrocytes and locally-derived non-angiogenic cells of neuroectodermal (pial) origin.

1. The chromatography of rat brain enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) reveals three distinet components. One of these appears to be isoenzyme 1 (alpha alpha) but isoenzymes 2 (alpha beta) and 3 (beta beta) are absent. 2. The most acidic form of brain enolase was partially purified and an antiserum raised against it in the chicken. 3. a combination of chromatographic and immunological studies showed that a third type of subunit (gamma) is present in the brain giving rise to two further isoenzymes (alpha gamma and gamma gamma). 4. Developmental studies on the brain enzyme show an increase in total enolase activity from foetal life to maturity and a concurrent rise in the proportion of brain specific dimers. 5. It is therefore concluded that there are three genetic loci alpha, beta and gamma, coding for the enolase isoenzymes of rat tissues.

All of 12 patients with Duchenne muscular dystrophy manifested raised activity of serum enolase but only 10 showed elevation of immunologically reacting muscle enolase (IRM-enolase). Total enolase was raised in 4 of 12 mothers and one of 8 sisters of the affected subjects but IRM-enolase was elevated in only one mother. These results are consistent with the view that enolase, unlike creatine phosphokinase which showed much more pronounced increase in the serum of patients and their female relatives, is associated with a structural component of the muscle cell distinct from the presumed site of molecular abnormality in this disease.

1. Hybridization studies have been carried out in vitro using mixtures of partially purified isoenzymes 1 and 3 of rat enolase (2-phospho-D-glycerate hydrolyase, EC 4.1.1.11). Immunological methods were used to demonstrate the formation of the hybrid. 2. Immunological analysis of the elution peaks from QAE-Sephadex chromatography of heart enolase indicates the occurrence in vivo of the hybrid enolase 2. 3. Developmental changes in the proportions of isoenzymes 1, 2 and 3 in heart and skeletal muscle of rat have been studied quantitatively. In both tissues isoenzyme 1 predominates in the foetus, but is partially replaced by 2 and 3 in adult heart and completely by 3 in the adult muscle. 4. Evidence is given of the binomial distribution of the proportions of the three isoenzymes in the developing heart. 5. Phylogenetic studies of the immunological properties of enolases from muscle, liver and heart have been carried out. 6. It is concluded that the three isoenzymes arise from two independent genetic loci and it is suggested that these evolved from a common ancestral gene 200-300 million years ago.

The utilisation of glucose, glutamine, acetoacetate and D-3-hydroxybutyrate were investigated over 72 h of incubation of rat splenic lymphocytes, with and without concanavalin A. Lymphocytes consumed both ketone bodies; acetoacetate was consumed preferentially. The ketone bodies reduced glucose consumption by 30-50%, but had little effect on lactate production. Glutamine uptake was concentration dependent up to 4 mM, and consumption was increased in the presence of concanavalin. Glutamine stimulated glucose consumption and lactate production in both resting and activated cells. Complete oxidation contributed 65% of glucose-derived ATP, but less than 40% of glutamine-derived ATP. Glutamine metabolism makes only a minor contribution to lymphocyte ATP generation.

Preincubation in assay mixture for 30 min at 37 degrees C of ATP citrate lyase from rat brain and liver results in 65-70% inhibition in the presence of 10 mM L-glutamate. This inhibition is specific since none of the known brain metabolites of glutamate shows this effect. ATP and ammonium sulphate-suspended, commercially purified malate dehydrogenase are both important in the generation of inhibition; citrate and NADH are not. The ATP citrate lyase activity in desalted crude extracts and 11% polyethylene glycol-precipitated fractions is inhibited but the enzyme purified by dye affinity chromatography is unaffected. Such purification reveals the presence of a factor responsible for the generation of the inhibition shown to be of Mr 380,000. These lines of evidence implicate endogenous glutamine synthetase, and the involvement of this enzyme is established by the use of its inhibitor L-methionine sulphoximine and by the addition of purified glutamine synthetase to restore the glutamate inhibition of purified ATP citrate lyase. The phenomenon probably arises from the production by glutamine synthetase of ADP, a known product inhibitor of ATP citrate lyase. Therefore contrary to previous reports elsewhere, L-glutamate has no role in the regulation of brain ATP citrate lyase and thus the supply of cytoplasmic acetyl groups for biosynthesis.