Structure and metabolism of multiple heparan sulphate proteoglycans synthesized by the isolated rat glomerulus.

Metabolism of biosynthetically [35S]sulphate-labelled heparan sulphate proteoglycan (HSPG) was studied in the isolated glomerulus. Chromatography and electrophoresis resolved HS into 5 components, designated HS1a, HS1b, and HS2 to HS4 in order of increasing Kd. Both HS1a (250 kDa) and HS1b (130 kDa) are present in the glomerular basement membrane and have glycosaminoglycan chains of 25-45 kDa. Chemical analysis of glycosaminoglycan chains indicated a similar content of 50% N-sulphation and 30% 6-O-sulphation on the hexosamine residues of all HSs, with the remaining 20% of sulphate likely at the 2-O-position of uronic acid residues. By pulse-chase analysis, the basement-membrane fraction was found to have a half-life of residency in the glomerulus of 37 h. Both HS1a and HS1b are mainly released intact into the medium and are not further broken down in that compartment. In contrast, HS2 is almost completely released into the medium immediately after synthesis and is not normally recovered from the tissue. It is a 90-kDa HSPG with a hydrophobic core protein and glycosaminoglycan chains similar in size to those of HS1. In addition to these larger PGs, HS3 and HS4 represent glycosaminoglycan chains with little or no core protein. HS1a, HS1b and HS2 were iodinated and deglycosylated. Each has a 30-kDa core protein in addition to 18 kDa of chondroitinase ABC- and nitrous-acid-resistant O-linked carbohydrate. This suggests the possibility of a single core protein with variable glycosylation and destination. HS1a has 5-6 glycosaminoglycan chains, HS1b 2-3 and HS2 1-2. We propose that basement-membrane HSPG (HS1a and HS1b) and a related, underglycosylated secreted HSPG (HS2) are the major HSPGs synthesized by the isolated glomerulus. Other molecular species may represent discrete steps in the turnover of basement-membrane HSPG.     

In vivo turnover of the basement membrane and other heparan sulfate proteoglycans of rat glomerulus

The metabolic turnover of rat glomerular proteoglycans in vivo was investigated. Newly synthesized proteoglycans were labeled during a 7-h period after injecting sodium [35S]sulfate intraperitoneally. At the end of the labeling period a chase dose of sodium sulfate was given. Subsequently at defined times (0-163 h) the kidneys were perfused in situ with 0.01% cetylpyridinium chloride in phosphate-buffered saline to maximize the recovery of 35S-proteoglycans. Glomeruli were isolated from the renal cortex and analyzed for 35S-proteoglycans by autoradiographic, biochemical, and immunochemical methods. Grain counting of autoradiographs revealed a complex turnover pattern of 35S-labeled macromolecules, commencing with a rapid phase followed by a slower phase. Biochemical analysis confirmed the biphasic pattern and showed that the total population of [35S]heparan sulfate proteoglycans had a metabolic half-life (t1/2) of 20 and 60 h in the early and late phases, respectively. Heparan sulfate proteoglycans accounted for 80% of total 35S-proteoglycans, the remainder being chondroitin/dermatan sulfate proteoglycans. Whole glomeruli were extracted with 4% 3-[(cholamidopropyl)dimethy-lammonio]-1-propanesulfonate-4 M guanidine hydrochloride, a procedure which solubilized greater than 95% of the 35S-labeled macromolecules. Of these 11-13% was immunoprecipitated by an antiserum against heparan sulfate proteoglycan which, in immunolocalization experiments, showed specificity for staining the basement membrane of rat glomeruli. Autoradiographic analysis showed that 18% of total radioactivity present at the end of the labeling period was associated with the glomerular basement membrane. The glomerular basement membrane [35S]heparan sulfate proteoglycans, identified by immunoprecipitation, have a very rapid turnover with an initial phase, t1/2 = 5 h, and a later phase t1/2 = 20 h.     

Synthesis by Schwann cells of basal lamina and membrane-associated heparan sulfate proteoglycans

Primary cultures that contain only Schwann cells and sensory nerve cells synthesize basal lamina. The assembly of this basal lamina appears to be essential for normal Schwann cell development. In this study, we demonstrate that Schwann cells synthesize two major heparan sulfate-containing proteoglycans. Both proteoglycans band in dissociative CsCl gradients at densities less than 1.4 g/ml, and therefore, presumably, have relatively low carbohydrate-to-protein ratios. The larger of these proteoglycans elutes from Sepharose CL-4B in 4 M guanidine hydrochloride (GuHCl) at a Kav of 0.21 and contains heparan sulfate and chondroitin sulfate chains of Mr 21,000 in a ratio of approximately 3:1. This proteoglycan is extracted from cultures by 4 M GuHCl but not Triton X-100 and accumulates only when Schwann cells are actively synthesizing basal lamina. The smaller proteoglycan elutes from Sepharose CL-4B at a Kav of 0.44 and contains heparan sulfate and chondroitin sulfate chains of Mr 18,000 in a ratio of approximately 4:1. This proteoglycan is extracted by 4 M GuHCl or by Triton X-100. The accumulation of this proteoglycan is independent of basal lamina production.     

Disulfide-bonded aggregates of heparan sulfate proteoglycans

Heparan sulfate proteoglycans have been isolated from Swiss mouse 3T3 cells by using two nondegradative techniques: extraction with 4 M guanidine or 2.5% 1-butanol. These proteoglycans were separated from copurifying chondroitin sulfate proteoglycans by using ion-exchange chromatography on DEAE-cellulose in the presence of 2 M urea. The purified heparan sulfate proteoglycans are substantially smaller, ca. Mr 20 000, than those isolated from these same cells with trypsin, ca. Mr 720 000 [Johnston, L.S., Keller, K. L., & Keller, J. M. (1979) Biochim. Biophys. Acta 583, 81-94]. However, all of the heparan sulfate proteoglycans extracted by these three methods contain similar glycosaminoglycan chains (Mr 7500) and are derived from the same pool of cell surface associated molecules. The trypsin-released heparan sulfate proteoglycan (ca. Mr 720 000) can be significantly reduced in size (ca. Mr 33 000) under strong denaturing conditions in the presence of the disulfide reducing agent dithiothreitol, which suggests that this form of the molecule is a disulfide-bonded aggregate. The heparan sulfate proteoglycan isolated from the medium also undergoes a significant size reduction in the presence of dithiothreitol, indicating that a similar aggregate is formed as part of the normal release of heparan sulfate proteoglycans into the medium. These results suggest that well-shielded disulfide bonds between individual heparan sulfate proteoglycan monomers may account for the large variation in sizes which has been reported for heparan sulfate proteoglycans isolated from a variety of cells and tissues with a variety of extraction procedures.     

Biology of cell surface heparan sulfate proteoglycans

The central question in cell biology is how cells detect, interact and respond to extracellular matrix. The cell surface molecules, which mediate this recognition, consist of a lipophilic membrane domain and an ectodomain binding matrix materials. One group of this kind of molecules is the cell surface heparan sulfate proteoglycans (HSPG). This review summarizes recent information obtained on the cell surface PG of mouse mammary epithelial cells. The glycosaminoglycan containing ectodomain of this PG binds with high affinity Type I, III and V collagen fibrils and the C-terminal heparin binding domain of fibronectin. The PG is mobile on the cell surface, but can be immobilised by ligand binding. At the same time the PG associates with cytoskeleton and links the epithelial cytoskeleton to extracellular matrix. Thus the PG can mediate the changes in the matrix into changes in cellular behaviour, often seen during the regulation of cell shape, proliferation and differentiation. The cell surface PG is also released from the cell surface by cleaving the matrix-binding ectodomain from the membrane domain. Because of the binding properties of the ectodomain, this shedding may provide a means by which epithelial cells loosen their association with the matrix and with other cells, e.g., during normal epithelial development and the invasion of carcinomas.     

Heterogeneity of heparan sulfate proteoglycans synthesized by PYS-2 cells

Antibodies to the basement membrane proteoglycan produced by the EHS tumor were used to immunoprecipitate [35S]sulfate-labeled protoglycans produced by PYS-2 cells. The immunoprecipitated proteoglycans were subsequently fractionated by CsCl density gradient centrifugation and Sepharose CL-4B chromatography. The culture medium contained a low-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.18, containing heparan sulfate side chains of Mr = 35-40,000. The medium also contained a high-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.23, containing heparan sulfate side chains of Mr = 30,000. The corresponding proteoglycans of the cell layer were all smaller than those in the medium. Since the antibodies used to precipitate those proteoglycans were directed against the protein core, this suggests that these proteoglycans share common antigenic features, and may be derived from a common precursor which undergoes modification by the removal of protein segments and a portion of each heparan sulfate chain.     

Heparanase uptake is mediated by cell membrane heparan sulfate proteoglycans

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intrachain sites, an activity that is strongly implicated in cell dissemination associated with metastasis and inflammation. In addition to its structural role in extracellular matrix assembly and integrity, HS sequesters a multitude of polypeptides that reside in the extracellular matrix as a reservoir. A variety of growth factors, cytokines, chemokines, and enzymes can be released by heparanase activity and profoundly affect cell and tissue function. Thus, heparanase bioavailability, accessibility, and activity should be kept tightly regulated. We provide evidence that HS is not only a substrate for, but also a regulator of, heparanase. Addition of heparin or xylosides to cell cultures resulted in a pronounced accumulation of, heparanase in the culture medium, whereas sodium chlorate had no such effect. Moreover, cellular uptake of heparanase was markedly reduced in HS-deficient CHO-745 mutant cells, heparan sulfate proteoglycan-deficient HT-29 colon cancer cells, and heparinase-treated cells. We also studied the heparanase biosynthetic route and found that the half-life of the active enzyme is approximately 30 h. This and previous localization studies suggest that heparanase resides in the endosomal/lysosomal compartment for a relatively long period of time and is likely to play a role in the normal turnover of HS. Co-localization studies and cell fractionation following heparanase addition have identified syndecan family members as candidate molecules responsible for heparanase uptake, providing an efficient mechanism that limits extracellular accumulation and function of heparanase.     

Developmental roles of heparan sulfate proteoglycans in Drosophila

The formation of complex patterns in multi-cellular organisms is regulated by a number of signaling pathways. In particular, the Wnt and Hedgehog (Hh) pathways have been identified as critical organizers of pattern in many tissues. Although extensive biochemical and genetic studies have elucidated the central components of the signal transduction pathways regulated by these secreted molecules, we still do not understand fully how they organize gradients of gene activities through field of cells. Studies in Drosophila have implicated a role for heparan sulfate proteoglycans (HSPGs) in regulating the signaling activities and distribution of both Wnt and Hh. Here we review these findings and discuss various models by which HSPGs regulate the distributions of Wnt and Hh morphogens. Published in 2003.     

Effect of chlorate on the sulfation of lipoprotein lipase and heparan sulfate proteoglycans. Sulfation of heparan sulfate proteoglycans affects lipoprotein lipase degradation

In avian-cultured adipocytes 76% of the newly synthesized lipoprotein lipase is degraded before release into the medium (Cupp, M., Bensadoun, A., and Melford, K. (1987) J. Biol. Chem. 262, 6383-6388). The same group (Cisar, L. A., Hoogewerf, A. J., Cupp, M., Rapport, C. A., and Bensadoun, A. (1989) J. Biol. Chem. 264, 1767-1774) has proposed that the interaction of lipoprotein lipase with a class of cell surface heparan sulfate proteoglycans is necessary for degradation to occur. To test further this hypothesis, the binding capacity of the plasma membrane for the lipase was decreased by inhibiting the sulfation of glycosaminoglycans with sodium chlorate, an inhibitor of sulfate adenyltransferase. Chlorate decreased sulfate incorporation into trypsin-releasable heparan sulfate proteoglycans to 20% of control levels. The amount of uronic acid in the trypsin-releasable heparan sulfate proteoglycans remained constant. Therefore, chlorate decreased sulfation density on heparan sulfate chains by approximately 5-fold. In the same fractions, chlorate increased the median heparan sulfate Mr measured on Sephacryl S-300. Chlorate decreased the maximum binding of 125I-lipoprotein lipase to adipocytes by 4-fold, but no significant effects on the affinity constants were observed. Chlorate increased lipoprotein lipase secretion in a dose-dependent relationship up to 30 mM. Utilizing a pulse-chase protocol, it was shown that lipase synthesis in control and chlorate-treated cells was not significantly different and that the increased secretion could be accounted for by a decreased lipoprotein lipase degradation rate. In control cells 77 +/- 11% of the synthesized enzyme was degraded whereas in chlorate-treated cells degradation was reduced to 42 +/- 9% of the synthesized amount. The present study shows that decreased sulfation of heparan sulfate proteoglycans decreases the maximum binding of the lipase for the adipocyte cell surface. Consistent with the model that binding of lipoprotein lipase to cell surface heparan sulfate is required for lipase degradation, degradation is reduced in chlorate-treated cultures. In this report it is also shown that chlorate inhibits lipoprotein lipase sulfation and that desulfation of the enzyme has no effect on its catalytic efficiency or on its binding to cultured adipocytes.     

Chondroitin sulfate and heparan sulfate proteoglycans of PC12 pheochromocytoma cells

We have isolated and characterized the cell-associated and secreted proteoglycans synthesized by a clonal line of rat adrenal medullary PC12 pheochromocytoma cells, which have been extensively employed for the study of a wide variety of neurobiological processes. Chondroitin sulfate accounts for 70-80% of the [35S] sulfate-labeled proteoglycans present in PC12 cells and secreted into the medium. Two major chondroitin sulfate proteoglycans were detected with molecular sizes of 45,000-100,000 and 120,000-190,000, comprising 14- and 105-kDa core proteins and one or two chondroitin sulfate chains with an average molecular size of 34 kDa. In contrast to the chondroitin sulfate proteoglycans, one major heparan sulfate proteoglycan accounts for most of the remaining 20-30% of the [35S] sulfate-labeled proteoglycans present in the PC12 cells and medium. It has a molecular size of 95,000-170,000, comprising a 65-kDa core protein and two to six 16-kDa heparan sulfate chains. Both the chondroitin sulfate and heparan sulfate proteoglycans also contain O-glycosidically linked oligosaccharides (25-28% of the total oligosaccharides) and predominantly tri- and tetraantennary N-glycosidic oligosaccharides. Proteoglycans produced by the original clone of PC12 cells were compared with those of two other PC12 cell lines (B2 and F3) that differ from the original clone in morphology, adhesive properties, and response to nerve growth factor. Although the F3 cells (a mutant line derived from B2 and reported to lack a cell surface heparan sulfate proteoglycan) do not contain a large molecular size heparan sulfate proteoglycan species, there was no significant difference between the B2 and F3 cells in the percentage of total heparan sulfate released by mild trypsinization, and both the B2 and F3 cells synthesized cell-associated and secreted chondroitin sulfate and heparan sulfate proteoglycans having properties very similar to those of the original PC12 cell line but with a reversed ratio (35:65) of chondroitin sulfate to heparan sulfate.     

Heparanases: endoglycosidases that degrade heparan sulfate proteoglycans.

Heparanases are endoglycosidases that cleave the heparan sulfate glycosaminoglycans from proteoglycan core proteins and degrade them to small oligosaccharides. Inside cells, these enzymes are important for the normal catabolism of heparan sulfate proteoglycans (HSPGs), generating glycosaminoglycan fragments that are then transported to lysosomes and completely degraded. When secreted, heparanases are thought to degrade basement membrane HSPGs at sites of injury or inflammation, allowing extravasion of immune cells into nonvascular spaces and releasing factors that regulate cell proliferation and angiogenesis. Heparanases have been described in a wide variety of tissues and cells, but because of difficulties in developing simple assays to follow activity, very little has been known about enzyme diversity until recently. Within the last 10 years, heparanases have been purified from platelets, placenta, and Chinese hamster ovary cells. Characterization of the enzymes suggests there may be a family of heparanase proteins with different substrate specificities and potential functions.     

Cathepsin X binds to cell surface heparan sulfate proteoglycans

Glycosaminoglycans have been shown to be important regulators of activity of several papain-like cathepsins. Binding of glycosaminoglycans to cathepsins thus directly affects catalytic activity, stability or the rate of autocatalytic activation of cathepsins. The interaction between cathepsin X and heparin has been revealed by affinity chromatography using heparin-Sepharose. Conformational changes were observed to accompany heparin-cathepsin X interaction by far UV-circular dichroism at both acidic (4.5) and neutral (7.4) pH. These conformational changes promoted a 4-fold increase in the dissociation constant of the enzyme-substrate interaction and increased 2.6-fold the kcat value also. The interaction between cathepsin X and heparin or heparan sulfate is specific since dermatan sulfate, chondroitin sulfate, and hyaluronic acid had no effect on the cathepsin X activity. Using flow cytometry cathepsin X was shown to bind cell surface heparan sulfate proteoglycans in wild-type CHO cells but not in CHO-745 cells, which are deficient in glycosaminoglycan synthesis. Moreover, fluorescently labeled cathepsin X was shown by confocal microscopy to be endocytosed by wild-type CHO cells, but not by CHO-745 cells. These results demonstrate the existence of an endocytosis mechanism of cathepsin X by the CHO cells dependent on heparan sulfate proteoglycans present at the cell surface, thus strongly suggesting that heparan sulfate proteoglycans can regulate the cellular trafficking and the enzymatic activity of cathepsin X.     

The critical interaction of the metallopeptidase PHEX with heparan sulfate proteoglycans

The PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) identified as a mutated gene in patients with X-linked hypophosphatemia (XLH), encodes a protein (PHEX) that shows striking homologies to members of the M13 family of zinc metallopeptidases. In the present work the interaction of glycosaminoglycans with PHEX has been investigated by affinity chromatography, circular dichroism, protein intrinsic fluorescence analysis, hydrolysis of FRET substrates flow cytometry and confocal microscopy. PHEX was eluted from a heparin-Sepharose chromatography column at 0.8 M NaCl showing a strong interaction with heparin. Circular dichroism spectra and intrinsic fluorescence analysis showed that PHEX is protected by glycosaminoglycans against thermal denaturation. Heparin, heparan sulfate and chondroitin sulfate inhibited PHEX catalytic activity, however among them, heparin presented the highest inhibitory activity (K_i = 2.5 ± 0.2 nM). Flow cytometry analysis showed that PHEX conjugated to Alexa Fluor 488 binds to the cell surface of CHO-K1, but did not bind to glycosaminoglycans defective cells CHO-745. Endogenous PHEX was detected at the cell surface of CHO-K1 colocalized with heparan sulfate proteoglycans, but was not found at the cell surface of glycosaminoglycans defective cells CHO-745. In permeabilized cells, PHEX was detected in endoplasmic reticulum of both cells. In addition, we observed that PHEX colocalizes with heparan sulfate at the cell surface of osteoblasts. This is the first report that the metallopeptidase PHEX is a heparin binding protein and that the interaction with GAGs modulates its enzymatic activity, protein stability and cellular trafficking.     

Functions of heparan sulfate proteoglycans in cell signaling during development

Heparan sulfate proteoglycans (HSPGs) are cell-surface and extracellular matrix macromolecules that are composed of a core protein decorated with covalently linked glycosaminoglycan (GAG) chains. In vitro studies have demonstrated the roles of these molecules in many cellular functions, and recent in vivo studies have begun to clarify their essential functions in development. In particular, HSPGs play crucial roles in regulating key developmental signaling pathways, such as the Wnt, Hedgehog, transforming growth factor-beta, and fibroblast growth factor pathways. This review highlights recent findings regarding the functions of HSPGs in these signaling pathways during development.     

Anti-clotting activity of endothelial cell cultures and heparan sulfate proteoglycans

A photoaffinity probe for the vitamin D-dependent chick intestinal calcium binding protein (CaBP) has been prepared by conjugation of methyl-4-azidobenzoimidate (MABI) to lactoperoxidase-¹²⁵I-iodinated CaBP to yield ¹²⁵I-CaBP-MABI: [3 moles MABI per mole CaBP]. After incubation $\text{in}\text{vitro}$ of ¹²⁵I-CaBP-MABI (28,000 daltons) in model systems with bovine intestinal alkaline phosphatase (AP) (67,000 daltons), a UV light-dependent crosslinking occurred to yield a conjugate with a molecular weight of 95,000 (by SDS-gel electrophoresis); no crosslinking occurred with $\text{E.}\text{coli}$ alkaline phosphatase. The formation of the ¹²⁵I-CaBP-MABI-AP was found to occur only in the presence of calcium.     

Anchorage of collagen-tailed acetylcholinesterase to the extracellular matrix is mediated by heparan sulfate proteoglycans

Heparan sulfate and heparin, two sulfated glycosaminoglycans (GAGs), extracted collagen-tailed acetylcholinesterase (AChE) from the extracellular matrix (ECM) of the electric organ of Discopyge tschudii. The effect of heparan sulfate and heparin was abolished by protamine; other GAGs could not extract the esterase. The solubilization of the asymmetric AChE apparently occurs through the formation of a soluble AChE-GAG complex of 30S. Heparitinase treatment but not chondroitinase ABC treatment of the ECM released asymmetric AChE forms. This provides direct evidence for the vivo interaction between asymmetric AChE and heparan sulfate residues of the ECM. Biochemical analysis of the electric organ ECM showed that sulfated GAGs bound to proteoglycans account for 5% of the total basal lamina. Approximately 20% of the total GAGs were susceptible to heparitinase or nitrous acid oxidation which degrades specifically heparan sulfates, and approximately 80% were susceptible to digestion with chondroitinase ABC, which degrades chondroitin-4 and -6 sulfates and dermatan sulfate. Our experiments provide evidence that asymmetric AChE and carbohydrate components of proteoglycans are associated in the ECM; they also indicate that a heparan sulfate proteoglycan is involved in the anchorage of the collagen-tailed AChE to the synaptic basal lamina.