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Increased deposition of specific extracellular matrix (ECM) components is a characteristic of insulin-resistant skeletal muscle. Hyaluronan (HA) is a major constituent of the ECM. The hypotheses that 1) HA content is increased in the ECM of insulin-resistant skeletal muscle and 2) reduction of HA in the muscle ECM by long-acting pegylated human recombinant PH20 hyaluronidase (PEGPH20) reverses high-fat (HF) diet-induced muscle insulin resistance were tested. We show that muscle HA was increased in HF diet-induced obese (DIO) mice and that treatment of PEGPH20, which dose-dependently reduced HA in muscle ECM, decreased fat mass, adipocyte size, and hepatic and muscle insulin resistance in DIO mice at 10 mg/kg. Reduced muscle insulin resistance was associated with increased insulin signaling, muscle vascularization, and percent cardiac output to muscle rather than insulin sensitization of muscle per se. Dose-response studies revealed that PEGPH20 dose-dependently increased insulin sensitivity in DIO mice with a minimally effective dose of 0.01 mg/kg. PEGPH20 at doses of 0.1 and 1 mg/kg reduced muscle HA to levels seen in chow-fed mice, decreased fat mass, and increased muscle glucose uptake. These findings suggest that ECM HA is a target for treatment of insulin resistance.

Conditionally active proteins regulated by a physiological parameter represent a potential new class of protein therapeutics. By systematically creating point mutations in the catalytic and linker domains of human MMP-1, we generated a protein library amenable to physiological parameter-based screening. Mutants screened for temperature-sensitive activity had mutations clustered at or near amino acids critical for metal binding. One mutant, GVSK (Gly(159) to Val, Ser(208) to Lys), contains mutations in regions of the catalytic domain involved in calcium and zinc binding. The in vitro activity of GVSK at 37 °C in high Ca(2+) (10 mm) was comparable with MMP-1 (wild type), but in low Ca(2+) (1 mm), there was an over 10-fold loss in activity despite having similar kinetic parameters. Activity decreased over 50% within 15 min and correlated with the degradation of the activated protein, suggesting that GVSK was unstable in low Ca(2+). Varying the concentration of Zn(2+) had no effect on GVSK activity in vitro. As compared with MMP-1, GVSK degraded soluble collagen I at the high but not the low Ca(2+) concentration. In vivo, MMP-1 and GVSK degraded collagen I when perfused in Zucker rat ventral skin and formed higher molecular weight complexes with α2-macroglobulin, an inhibitor of MMPs. In vitro and in vivo complex formation and subsequent enzyme inactivation occurred faster with GVSK, especially at the low Ca(2+) concentration. These data suggest that the activity of the human MMP-1 mutant GVSK can be regulated by Ca(2+) both in vitro and in vivo and may represent a novel approach to engineering matrix-remodeling enzymes for therapeutic applications.

The metabolism of hyaluronan (HA), especially its catabolism, is still far from being elucidated. Although several studies suggest that HA is degraded locally in tissues and through the lymphatic or circulatory systems, much needs to be learned about the enzymes, receptors and cell types that support this dynamic process. In the current work, the clearance of exogenously administered HA was examined in a C57BL/6 mouse model. Hyaluronidase-sensitive fluorescein-labeled 1.2MDa hyaluronan (flHA) was administered either intravenously (i.v.) or subcutaneously (s.c.) into wild type C57BL/6 mice. Plasma was sampled for pharmacokinetic analysis and tissues were harvested for histological examination of the cell types responsible for uptake using immunofluorescent localization and for size exclusion chromatography analysis. We observed that flHA could be degraded locally in the skin or be taken up by sinusoidal cells in lymph nodes, liver and spleen. I.v. administration of flHA revealed non-linear Michaelis-Menten pharmacokinetics compatible with a saturable, receptor-mediated clearance system (K(m)=11.6μg/ml±46.0%, V(max)=1.69μg/ml/min±59.7%). Through a combination of immunofluorescence microscopy, pharmacokinetic, and chromatographic analyses of labeled substrate in vivo, our results shed additional light on the mechanisms by which HA is catabolized in mammals, and serve as a basis for future studies., (Copyright © 2011 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Hyaluronan (HA) is a glycosaminoglycan polymer that often accumulates in malignancy. Megadalton complexes of HA with proteoglycans create a hydrated connective tissue matrix, which may play an important role in tumor stroma formation. Through its colloid osmotic effects, HA complexes contribute to tumor interstitial fluid pressure, limiting the effect of therapeutic molecules on malignant cells. The therapeutic potential of enzymatic remodeling of the tumor microenvironment through HA depletion was initially investigated using a recombinant human HA-degrading enzyme, rHuPH20, which removed HA-dependent tumor cell extracellular matrices in vitro. However, rHuPH20 showed a short serum half-life (t(1/2) < 3 minutes), making depletion of tumor HA in vivo impractical. A pegylated variant of rHuPH20, PEGPH20, was therefore evaluated. Pegylation improved serum half-life (t(1/2) = 10.3 hours), making it feasible to probe the effects of sustained HA depletion on tumor physiology. In high-HA prostate PC3 tumors, i.v. administration of PEGPH20 depleted tumor HA, decreased tumor interstitial fluid pressure by 84%, decreased water content by 7%, decompressed tumor vessels, and increased tumor vascular area >3-fold. Following repeat PEGPH20 administration, tumor growth was significantly inhibited (tumor growth inhibition, 70%). Furthermore, PEGPH20 enhanced both docetaxel and liposomal doxorubicin activity in PC3 tumors (P < 0.05) but did not significantly improve the activity of docetaxel in low-HA prostate DU145 tumors. The ability of PEGPH20 to enhance chemotherapy efficacy is likely due to increased drug perfusion combined with other tumor structural changes. These results support enzymatic remodeling of the tumor stroma with PEGPH20 to treat tumors characterized by the accumulation of HA., (©2010 AACR.)

Subcutaneously injected therapeutics must pass through the interstitial matrix of the skin in order to reach their intended targets. This complex, three-dimensional structure limits the type and quantity of drugs that can be administered by local injection. Here we found that depolymerization of the viscoelastic component of the interstitial matrix in animal models with a highly purified recombinant human hyaluronidase enzyme (rHuPH20) increased the dispersion of locally injected drugs, across a broad range of molecular weights without tissue distortion. rHuPH20 increased infusion rates and the pattern and extent of appearance of locally injected drugs in systemic blood. In particular, rHuPH20 changed the pharmacokinetic profiles and significantly augmented the absolute bioavailability of locally injected large protein therapeutics. Importantly, within 24 h of injection, the interstitial viscoelastic barriers were restored without histologic alterations or signs of inflammation. rHuPH20 may function as an interstitial delivery enhancing agent capable of increasing the dispersion and bioavailability of coinjected drugs that may enable subcutaneous administration of therapeutics and replace intravenous delivery.

Mouse sperm recognize and bind to ZP3, one of three glycoproteins in the egg's zona pellucida. A mouse sperm protein, sp56, was identified that has the characteristics expected of the sperm protein responsible for recognition of ZP3. The complementary DNA encoding sp56 was isolated, and its primary sequence indicates that sp56 is a member of a superfamily of protein receptors. It was shown that sp56 expression is restricted to mouse spermatids and that the presence or absence of sp56 on sperm from different species accounts for species specificity of sperm-egg recognition in mice.

Recognition between mammalian gametes occurs when the plasma membrane of the sperm head binds to the zona pellucida (ZP), an extracellular coat surrounding eggs. ZP3, one of three glycoproteins in the ZP, is the egg protein recognized by sperm. A mouse sperm surface protein, sp56 (M(r) = 56,000), has been identified on the basis of its specific affinity for ZP3 (Bleil, J. D., and P. M. Wassarman. 1990. Proc. Natl. Acad. Sci. USA. 87:5563-5567). Studies presented here were designed to characterize mouse sperm sp56 and to further test whether or not this protein specifically recognizes ZP3. sp56 was purified by both ZP3 affinity chromatography and by ion exchange chromatography followed by size-exclusion chromatography. The purified native protein eluted from size-exclusion columns as a homomultimer (M(r) approximately 110,000). Each monomer of the protein contains intramolecular disulfide bonds, consistent with its extracellular location. Immunohistochemical and immunoblotting studies, using monoclonal antibodies, demonstrated that sp56 is a peripheral membrane protein located on the outer surface of the sperm head plasma membrane, precisely where sperm bind ZP3. Results of crosslinking experiments demonstrated that the ZP3 oligosaccharide recognized by sperm has specific affinity for sp56. Collectively, these results suggest that sp56 may be the sperm protein responsible for sperm-egg recognition in the mouse.

The neural-specific calmodulin-sensitive adenylyl cyclase (type I), which was first cloned from bovine brain, has been implicated in learning and memory. The objective of this study was to clone and determine the chromosomal localization of human fetal brain type I adenylyl cyclase. A 3.8-kb cDNA clone was isolated that contained sequence coinciding with the 3' end 2553 nucleotides of the bovine open reading frame. This clone shows 87% nucleotide and 92% translated amino acid sequence identity to the bovine clone. The most significant sequence differences were in the carboxy-terminal 100 amino acid residues. This region contains one of several possible calmodulin binding domains and the only putative cAMP-dependent protein kinase A phosphorylation site. A chimera was constructed that contained the 5' half of the bovine type I adenylyl cyclase and the 3' half of the human type I adenylyl cyclase. The activity of the chimeric gene product and its sensitivity to calmodulin and calcium were indistinguishable from those of the bovine type I adenylyl cyclase. In situ hybridization was used to localize the human type I adenylyl cyclase gene to the proximal portion of the short arm of chromosome 7.

Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.

Calmodulin (CaM) affinity chromatography of a detergent extract of sea urchin sperm yielded approximately 20 major proteins. One of these proteins, of Mr 190,000, was purified and used to immunize rabbits. After absorption with living sperm, the serum reacted monospecifically on one- and two-dimensional Western immunoblots with the Mr 190,000 protein. The anti-190-kD serum inhibited 94% of the adenylate cyclase (AC) activity of the CaM eluate. An immunoaffinity column removed 95% of the AC activity, and the purified (but inactive) Mr 190,000 protein was eluted from the column. The antiserum also inhibited 23% of the activity of bovine brain CaM-sensitive AC and 90% of the activity of horse sperm CaM-sensitive AC. These data support the hypothesis that the Mr 190,000 protein is sea urchin sperm AC. Although this AC bound to CaM, it was not possible to demonstrate directly a Ca2+ or CaM sensitivity. However, two CaM antagonists, calmidazolium and chlorpromazine, both inhibited AC activity, and the inhibition was released by added CaM, suggesting the possibility of regulation of this AC by CaM. Indirect immunofluorescence showed the Mr 190,000 protein to be highly concentrated on only the proximal half of the sea urchin sperm flagellum. This asymmetric localization of AC may be important to its function in flagellar motility. This is the first report of the identification of an AC from animal spermatozoa.