Immunological characterization of basement membrane types of heparan sulfate proteoglycan

Antibodies were raised against a small high-density and a large low-density form of heparan sulfate proteoglycan from a basement membrane-producing mouse tumor and were characterized by radioimmunoassays, immunoprecipitation and immunohistological methods. Antigenicity was due to the protein cores and included epitopes unique to the low density form as well as some shared by both proteoglycans. The antibodies did not cross-react with other basement membrane proteins or with chondroitin sulfate proteoglycans from interstitial connective tissues. The heparan sulfate proteoglycans occurred ubiquitously in embryonic and adult basement membranes and could be initially detected at the 2-4 cell stage of mouse embryonic development. Low levels were also found in serum. Biosynthetic studies demonstrated identical or similar proteoglycans in cultures of normal and carcinoembryonic cells and in organ cultures of fetal tissues. They could be distinguished from liver cell membrane heparan sulfate proteoglycan, indicating that the basement membrane types of proteoglycans represent a unique class of extracellular matrix proteins.     

Molecular cloning of amphiglycan, a novel integral membrane heparan sulfate proteoglycan expressed by epithelial and fibroblastic cells

We have synthesized an antisense oligonucleotide primer that matches a supposedly conserved sequence in messages for heparan sulfate proteoglycans with transmembrane orientations. With the aid of this primer we have amplified partial and selected full-length copies of a message from human lung fibroblasts that codes for a novel integral membrane heparan sulfate proteoglycan. The encoded protein is 198 amino-acids long, with discrete cytoplasmic, transmembrane, and amino-terminal extracellular domains. Except for the sequences that represent putative heparan sulfate chain attachment sites, the extracellular domain of this protein has a unique structure. The transmembrane and cytoplasmic domains, in contrast, are highly similar to the corresponding domains of fibroglycan and syndecan, the two cell surface proteoglycans that figured as models for the design of the antisense primer. This similarity includes the conservation of four tyrosine residues, one immediately in front of the stop transfer sequence and three in the cytoplasmic segment, and of the most proximal and most distal cytoplasmic sequences. The cDNA detects a single 2.6-kb message in cultured human lung fibroblasts and in a variety of human epithelial and fibroblastic cell lines. Polyclonal and monoclonal antibodies raised against the encoded peptide after expression as a beta-galactosidase fusion protein react with the 35-kD coreprotein of a cell surface heparan sulfate proteoglycan of human lung fibroblasts and decorate the surface of many cell types. We propose to name this proteoglycan "amphiglycan" (from the Greek words amphi, "around, on both sides of" and amphoo, "both") referring to its domain structure which extends on both sides of the plasmamembrane, and to its localization around cells of both epithelial and fibroblastic origin.     

Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells

Evidence suggests that endothelial cell layer heparan sulfate proteoglycans include a variety of different sized molecules which most likely contain different protein cores. In the present report, approximately half of endothelial cell surface associated heparan sulfate proteoglycan is shown to be releasable with soluble heparin. The remaining cell surface heparan sulfate proteoglycan, as well as extracellular matrix heparan sulfate proteoglycan, cannot be removed from the cells with heparin. The heparin nonreleasable cell surface proteoglycan can be released by membrane disrupting agents and is able to intercalate into liposomes. When the heparin releasable and nonreleasable cell surface heparan sulfate proteoglycans are compared, differences in proteoglycan size are also evident. Furthermore, the intact heparin releasable heparan sulfate proteoglycan is closer in size to proteoglycans isolated from the extracellular matrix and from growth medium than to that which is heparin nonreleasable. These data indicate that cultured porcine aortic endothelial cells contain at least two distinct types of cell surface heparan sulfate proteoglycans, one of which appears to be associated with the cells through its glycosaminoglycan chains. The other (which is more tightly associated) is probably linked via a membrane intercalated protein core.Abbreviations ECM extracellular matrix - HSPG heparan sulfate proteoglycan - PAE porcine aortic endothelial - PBS phosphate buffered saline     

Differential expression of cell surface heparan sulfate proteoglycans in human mammary epithelial cells and lung fibroblasts.

Treating the liposome-intercalatable heparan sulfate proteoglycans from human lung fibroblasts and mammary epithelial cells with heparitinase and chondroitinase ABC revealed different core protein patterns in the two cell types. Lung fibroblasts expressed heparan sulfate proteoglycans with core proteins of approximately 35, 48/90 (fibroglycan), 64 (glypican), and 125 kDa and traces of a hybrid proteoglycan which carried both heparan sulfate and chondroitin sulfate chains. The mammary epithelial cells, in contrast, expressed large amounts of a hybrid proteoglycan and heparan sulfate proteoglycans with core proteins of approximately 35 and 64 kDa, but the fibroglycan and 125-kDa cores were not detectable in these cells. Phosphatidylinositol-specific phospholipase C and monoclonal antibody (mAb) S1 identified the 64-kDa core proteins as glypican, whereas mAb 2E9, which also reacted with proteoglycan from mouse mammary epithelial cells, tentatively identified the hybrid proteoglycans as syndecan. The expression of syndecan in lung fibroblasts was confirmed by amplifying syndecan cDNA sequences from fibroblastic mRNA extracts and demonstrating the cross-reactivity of the encoded recombinant core protein with mAb 2E9. Northern blots failed to detect a message for fibroglycan in the mammary epithelial cells and in several other epithelial cell lines tested, while confirming the expression of both glypican and syndecan in these cells. Confluent fibroblasts expressed higher levels of syndecan mRNA than exponentially growing fibroblasts, but these levels remained lower than observed in epithelial cells. These data formally identify one of the cell surface proteoglycans of human lung fibroblasts as syndecan and indicate that the expression of the cell surface proteoglycans varies in different cell types and under different culture conditions.     

Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line

A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl- Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation.     

Regulated changes in chondroitin sulfation during embryogenesis: an immunohistochemical approach

Chondroitin sulfate proteoglycans, which represent the main class of nonfibrous macromolecules found in the extracellular matrix of connective tissues, have been implicated in the control of a variety of cell activities during ontogenesis. The respective contributions of the chondroitin sulfate chains and of the protein moiety of the proteoglycan in morphogenesis and cytodifferentiation are not known. In this context, monoclonal antibodies identifying specific chondroitin sulfate chains are interesting new tools. A panel of well characterized monoclonal antibodies recognizing distinct epitopes present only in chondroitin sulfate chains was used in conjunction with immunohistochemical techniques for the purpose of identifying and mapping chondroitin sulfate isoforms during development in the mouse and rat fetus. Expression of chondroitin sulfate isoforms occurred in the tissues according to specific spatio-temporal patterns, suggesting that chondroitin sulfates differing in sulfation position and degree perform distinct functions in development.     

The dynamics of compartmentalization of embryonic muscle by extracellular matrix molecules

In order to delineate the role of proteoglycans in muscle development, the immunohistological localization of glycosaminoglycans and proteoglycan core proteins was studied in embryonic chick leg at Hamburger-Hamilton stages (St.) 36, 39, 43, and 46, and at 2 weeks posthatching. A specific anatomical landmark was chosen (the junction between the pars pelvica and the pars accessoria of the flexor cruris lateralis muscle) in order to ensure the study of anatomically equivalent sites. Frozen cross sections were immunostained with monoclonal antibodies to chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, and keratan sulfate glycosaminoglycans; to the core proteins of muscle/mesenchymal chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, and basement membrane heparan sulfate proteoglycan; and to laminin and tenascin. Extracellular matrix zones corresponding to the endomysium, perimysium, epimysium, basement membrane, and myotendinous junction each show characteristic immunostaining patterns from St. 36 to St. 46 and have unique matrix compositions by St. 46. In some cases, there is a sequential or coordinate expression of epitopes, first in the epimysium, then the perimysium, and last in the endomysium. Dermatan sulfate proteoglycan is detected in the epimysium at St. 36, in the perimysium at St. 39 (there is no perimysium structure at St. 36), and is not detected in the endomysium until St. 43. A putative mesenchymal proteoglycan core protein (reactive to the monoclonal antibody MY-174) is detected at St. 39 in both epimysium and perimysium, but is not detected in the endomysium until St. 43. Keratan sulfate antibody immunostains epimysium at St. 39 and perimysium at St. 46, but is never detected in the endomysium. Some epitopes are expressed independently in each of the extracellular matrix zones: antibody to tenascin stains only a subset of the epimysium, at the myotendinous junction; and heparan sulfate proteoglycan and laminin are detected only in the endomysium. Between St. 36 and St. 39, the muscle/MY-174-reactive proteoglycan core protein staining decreases in intensity in the endomysium and becomes positive in the epimysium and perimysium. An inverse relationship is found between (1) the disappearance of muscle/MY-174-reactive proteoglycan core protein staining at the surface of myotubes from St. 36 to St. 39 and (2) the infiltration of laminin and heparan sulfate proteoglycan staining encompassing groups of myotubes (St. 36) to circumferential staining of all myotubes (St. 39).(ABSTRACT TRUNCATED AT 400 WORDS)     

Basement membrane-like matrix of teratocarcinoma-derived endodermal cells: presence of laminin and heparan sulfate in the matrix at points of attachment to cells

Teratocarcinoma-derived endodermal PYS-2 cells are known to synthesize an extracellular matrix containing the basement membrane molecules laminin, type IV collagen, and heparan sulfate proteoglycan as major constituents (I. Leivo, K. Alitalo, L. Risteli, A. Vaheri, R. Timpl, J. Wartiovaara, Exp Cell Res 137:15-23, 1982). Immunoferritin techniques with specific antibodies were used in the present study to define the ultrastructural localization of the above constituents in the fibrillar network. Laminin was detected in matrix network adjacent to the basal cell membrane and in protruding matrix fibrils that connect the matrix to the cell membrane. Ruthenium red-stainable heparinase-sensitive 10- to 20-nm particles were often present at the junction of the attachment fibrils and the matrix network, or along the attachment fibrils. A corresponding distribution of ferritin label was observed for basement membrane heparan sulfate proteoglycan. Type IV collagen was found in the matrix network but not in the attachment fibrils. The results suggest that the PYS-2 cells are connected to their pericellular matrix by fibrils containing laminin associated with heparan sulfate-containing particles. These results may also have relevance for the attachment of epithelial cells to basement membranes.     

Immunochemical characterization and ultrastructural localization of chondroitin sulfates and keratan sulfate in embryonic chick bone marrow

Summary Monoclonal antibodies directed against specific carbohydrate epitopes on chondroitin 4-/dermatan sulfate, chondroitin 6-sulfate, keratan sulfate, and a monoclonal antibody directed against the hyaluronate binding region were used to characterize proteoglycans extracted from embryonic chick bone marrow. About half of the proteoglycans separate into the high density fraction on a CsCl gradient. Glycosaminoglycan-specific antibodies recognize proteoglycans from all fractions; this includes an antibody directed against keratan sulfate. Some proteoglycans, principally in the high buoyant density fraction, contain sites recognized by the antibody specific for the hyaluronate binding region. Within limits of detection, all core proteins belong to the high-molecular-weight category, with weights in excess of 212 kD. Antibodies directed against chondroitin 4-/dermatan sulfate and against keratan sulfate primarily bind to extracellular matrix material located in the extracellular spaces and to matrix elements in the pericellular regions of fibroblastic stromal cells. The antibody that recognizes chondroitin 6-sulfate binds to sites on surfaces of fibroblastic stromal cells and also to extracellular matrix material. Little or no antibody binding is detected on surfaces of granulocytic cells. These studies indicate that chondroitin sulfate and keratan sulfate chains are both present in the proteoglycan extract.     

Mouse mammary epithelial cells produce basement membrane and cell surface heparan sulfate proteoglycans containing distinct core proteins

Cultured mouse mammary (NMuMG) cells produce heparan sulfate-rich proteoglycans that are found at the cell surface, in the culture medium, and beneath the monolayer. The cell surface proteoglycan consists of a lipophilic membrane-associated domain and an extracellular domain, or ectodomain, that contains both heparan and chondroitin sulfate chains. During culture, the cells release into the medium a soluble proteoglycan that is indistinguishable from the ectodomain released from the cells by trypsin treatment. This medium ectodomain was isolated, purified, and used as an antigen to prepare an affinity-purified serum antibody from rabbits. The antibody recognizes polypeptide determinants on the core protein of the ectodomain of the cell surface proteoglycan. The reactivity of this antibody was compared with that of a serum antibody (BM-1) directed against the low density basement membrane proteoglycan of the Englebarth-Holm-Swarm tumor (Hassell, J. R., W. C. Leyshon, S. R. Ledbetter, B. Tyree, S. Suzuki, M. Kato, K. Kimata, and H. Kleinman. 1985. J. Biol. Chem. 250:8098-8105). The BM-1 antibody recognized a large, low density heparan sulfate-rich proteoglycan in the cells and in the basal extracellular materials beneath the monolayer where it accumulated in patchy deposits. The affinity-purified anti-ectodomain antibody recognized the cell surface proteoglycan on the cells, where it is seen on apical cell surfaces in subconfluent cultures and in fine filamentous arrays at the basal cell surface in confluent cultures, but detected no proteoglycan in the basal extracellular materials beneath the monolayer. The amino acid composition of the purified medium ectodomain was substantially different from that reported for the basement membrane proteoglycan. Thus, NMuMG cells produce at least two heparan sulfate-rich proteoglycans that contain distinct core proteins, a cell surface proteoglycan, and a basement membrane proteoglycan. In newborn mouse skin, these proteoglycans localize to distinct sites; the basement membrane proteoglycan is seen solely at the dermal-epidermal boundary and the cell surface proteoglycan is seen solely at the surfaces of keratinocytes in the basal, spinous, and granular cell layers. These results suggest that although heparan sulfate-rich proteoglycans may have similar glycosaminoglycan chains, they are sorted by the epithelial cells to different sites on the basis of differences in their core proteins.     

Spatial and temporal changes in the distribution of proteoglycans during avian neural crest development

In this study, we describe the distribution of various classes of proteoglycans and their potential matrix ligand, hyaluronan, during neural crest development in the trunk region of the chicken embryo. Different types of chondroitin and keratan sulfate proteoglycans were recognized using a panel of monoclonal antibodies produced against specific epitopes on their glycosaminoglycan chains. A heparan sulfate proteoglycan was identified by an antibody against its core protein. The distribution of hyaluronan was mapped using a biotinylated fragment that corresponds to the hyaluronan-binding region of cartilage proteoglycans. Four major patterns of proteoglycan immunoreactivity were observed. (1) Chondroitin-6-sulfate-rich proteoglycans and certain keratin sulfate proteoglycans were absent from regions containing migrating neural crest cells, but were present in interstitial matrices and basement membranes along prospective migratory pathways such as the ventral portion of the sclerotome. Although initially distributed uniformly along the rostrocaudal extent of the sclerotome, these proteoglycans became rearranged to the caudal portion of the sclerotome with progressive migration of neural crest cells through the rostral sclerotome and their aggregation into peripheral ganglia. (2) A subset of chondroitin/keratan sulfate proteoglycans bearing primarily unsulfated chondroitin chains was observed exclusively in regions where neural crest cells were absent or delayed from entering, such as the perinotochordal and subepidermal spaces. (3) A subset of chondroitin/keratan sulfate proteoglycans was restricted to the perinotochordal region and, following gangliogenesis, was arranged in a metameric pattern corresponding to the sites where presumptive vertebral arches form. (4) Certain keratan sulfate proteoglycans and a heparan sulfate proteoglycan were observed in basement membranes and in an interstitial matrix uniformly distributed along the rostrocaudal extent of the sclerotome. After gangliogenesis, the neural crest-derived dorsal root and sympathetic ganglia contained both these proteoglycan types, but were essentially free of other chondroitin/keratan-proteoglycan subsets. Hyaluronan generally colocalized with the first set of proteoglycans, but also was concentrated around migrating neural crest cells and was reduced in neural crest-derived ganglia. These observations demonstrate that proteoglycans have diverse and dynamic distributions during times of neural crest development and chondrogenesis of the presumptive vertebrae. In general, chondroitin/keratan sulfate proteoglycans are abundant in regions where neural crest cells are absent, and their segmental distribution inversely correlates with that of neural crest-derived ganglia.     

Heparan sulfate proteoglycans on the cell surface: versatile coordinators of cellular functions

Heparan sulfate proteoglycans are complex molecules composed of a core protein with covalently attached glycosaminoglycan chains. While the protein part determines localization of the proteoglycan on the cell surfaces or in the extracellular matrix, the glycosaminoglycan component, heparan sulfate, mediates interactions with a variety of extracellular ligands such as growth factors and adhesion molecules. Through these interactions, heparan sulfate proteoglycans participate in many events during cell adhesion, migration, proliferation and differentiation. We are determining the multitude of proteoglycan functions, as their intricate roles in many pathways are revealed. They act as coreceptors for growth factors, participate in signalling during cell adhesion, modulate the activity of a broad range of molecules, and partake in many developmental and pathological processes, including tumorigenesis and wound repair. This review concentrates on biological roles of cell surface heparan sulfate proteoglycans, namely syndecans and glypicans, and outlines the progress achieved during the last decade in unraveling the molecular interactions behind proteoglycan functions.     

Codistribution of heparan sulfate proteoglycan, laminin, and fibronectin in the extracellular matrix of normal rat kidney cells and their coordinate absence in transformed cells

We used antibodies raised against both a heparan sulfate proteoglycan purified from a mouse sarcoma and a chondroitin sulfate proteoglycan purified from a rat yolk sac carcinoma to study the appearance and distribution of proteoglycans in cultured cells. Normal rat kidney cells displayed a fibrillar network of immunoreactive material at the cell surface when stained with antibodies to heparan sulfate proteoglycan, while virally transformed rat kidney cells lacked such a surface network. Antibodies to chondroitin sulfate proteoglycan revealed a punctate pattern on the surface of both cell types. The distribution of these two proteoglycans was compared to that of fibronectin by double-labeling immunofluorescent staining. The heparan sulfate proteoglycan was found to codistribute with fibronectin, and fibronectin and laminin gave coincidental stainings. The distribution of chondroitin sulfate proteoglycan was not coincidental with that of fibronectin. Distinct fibers containing fibronectin but lacking chondroitin sulfate proteoglycan were observed. When the transformed cells were cultured in the presence of sodium butyrate, their morphology changed, and fibronectin, laminin, and heparan sulfate proteoglycan appeared at the cell surface in a pattern resembling that of normal cells. These results suggest that fibronectin, laminin, and heparan sulfate proteoglycan may be complexed at the cell surface. The proteoglycan may play a central role in assembly of such complexes since heparan sulfate has been shown to interact with both fibronectin and laminin.     

Developmental changes in heparan sulfate expression: in situ detection with mAbs

Two mAbs that are specific for heparan sulfate-related epitopes have been raised and used to analyze the cellular and tissular distribution of this glycosaminoglycan during development. mAb 10E4 reacts with an epitope that occurs in native heparan sulfate chains and that is destroyed by N-desulfation of the glycosaminoglycan. The antibody does not react with hyaluronate, chondroitin sulfate, or DNA, and reacts only poorly with heparin. The reactivity of proteoglycan extracts or tissue sections with the 10E4 antibody is completely abolished by heparitinase, but is only partially affected by heparinase. mAb 3G10, in contrast, reacts only with heparitinase-treated heparan sulfate chains, proteoglycans, or tissue sections. The 3G10 epitope is destroyed by treatment with mercuric acetate, which indicates that the desaturated uronate generated by the lyase is essential for the reactivity of the antibody. The 3G10 epitope is not generated by treating heparan sulfate proteoglycans with heparinase or chondroitin sulfate proteoglycans with chondroitin sulfate lyases, which indicates that the 3G10 antibody recognizes desaturated uronates that occur in specific structural contexts. The antibody 10E4 and, after heparitinase treatment, the antibody 3G10 decorate the surfaces of many cell types and the extracellular matrix in proximity of the cells, in particular, the basement membranes. The analysis of embryonic and adult tissues reveals important temporal and regional differences in the abundance of the 10E4 and 3G10 epitopes at these sites. Moreover, the staining pattern of the two antibodies is not always superimposable, which is indicative of regional differences in the exposure or structure of the tissular heparan sulfates. As a whole the results suggest that heparan sulfate abounds at sites of active morphogenesis and that the expression of this glycosaminoglycan is developmentally regulated.     

A Monoclonal Antibody which Recognizes a Glycosaminoglycan Epitope in Both Dermatan Sulfate and Chondroitin Sulfate Proteoglycans of Human Skin

Studies have been initiated to identify various cell surface and matrix components of normal human skin through the production and characterization of murine monoclonal antibodies. One such antibody, termed PG-4, identifies both cell surface and matrix antigens in extracts of human foetal and adult skin as the dermatan sulfate proteoglycans, decorin and biglycan, and the chondroitin sulfate proteoglycan versican. Treatment of proteoglycans with chondroitinases completely abolishes immunoreactivity for all of these antigens which suggests that the epitope resides within their glycosaminoglycan chains. Further evidence for the carbohydrate nature of the epitope derives from competition studies where protein-free chondroitin sulfate chains from shark cartilage react strongly; however, chondroitin sulfate chains from bovine tracheal cartilage fail to exhibit a significant reactivity, an indication that the epitope, although present in some chondroitin sulfate chains, does not consist of random chondroitin 4- or 6-sulfate disaccharides. The presence of the epitope on dermatan sulfate chains and on decorin was also demonstrated using competition assays. Thus, PG-4 belongs to a class of antibodies that recognize native epitopes located within glycosaminoglycan chains. It differs from previously described antibodies in this class in that it identifies both chondroitin sulfate and dermatan sulfate proteoglycans. These characteristics make PG-4 a useful monoclonal antibody probe to identify the total population of proteoglycans in human skin.     

Partial characterization of proteoglycans synthesized by human glomerular epithelial cells in culture

Confluent adult and fetal human glomerular epithelial cells were incubated for 24 h in the presence of [3H]-amino acids and [35S]sulfate. Two heparan-35SO4 proteoglycans were released into the culture medium. These 35S-labeled proteoglycans eluted as a single peak from anion exchange chromatographic columns, but were separable by gel filtration on Sepharose CL-6B columns. The larger heparan-35SO4 proteoglycan eluted with the column void volume and at a Kav of 0.26 from Sepharose CL-4B columns. The most abundant medium heparan-35SO4 proteoglycan was a high buoyant density proteoglycan similar in hydrodynamic size (Sepharose CL-6B Kav 0.23) to those previously described in glomerular basement membranes and isolated glomeruli. Heparan-35SO4 chains from both proteoglycans were 36 kDa. A smaller proportion of Sepharose CL-6B excluded dermatan-35SO4 proteoglycan was also synthesized by these cells. The predominant protein cores of both medium heparan-35SO4 proteoglycans were approximately 230 and 180 kDa. A hybrid chondroitin/dermatan-heparan-35SO4 proteoglycan with an 80-kDa protein core copurified with the smaller medium heparan-35SO4 proteoglycan. This 35S-labeled proteoglycan appeared as a diffuse, chondroitinase ABC sensitive 155-kDa fluorographic band in sodium dodecyl sulfate-polyacrylamide gels after the Sepharose CL-6B Kav 0.23 35S-labeled proteoglycan fraction was digested with heparitinase. The heparitinase generated heparan sulfate proteoglycan protein cores and the 155-kDa hybrid proteoglycan fragment had molecular weights similar to those previously identified in rat glomerular basement membrane and glomeruli using antibodies against a basement membrane tumor proteoglycan precursor (Klein et al. J. Cell Biol. 106, 963-970, 1988). Thus, human glomerular epithelial cells in culture are capable of synthesizing, processing, and releasing heparan sulfate proteoglycans which are similar to those synthesized in vivo and found in the glomerular basement membrane. These proteoglycans may belong to a family of related basement membrane proteoglycans.     

Energy of the low-lying excited levels for some reduced [4Fe-4S] ferredoxins, from the relaxation broadening of the E.P.R. signals

Two different forms of cell-associated [³⁵S]-heparan sulfate proteoglycans were identified in prelabeled cultured cells, including glial cells, endothelial cells and fibroblasts. One of them migrated characteristically in the excluded volume fraction in Sepharose CL-2B chromatography and flotated in CsCl density gradient centrifugation. Further, it showed affinity for a hydrophobic gel, Octyl-Sepharose. The molecular size was markedly reduced and the density elevated by treatment with detergent or lipid solvents. These findings indicate an admixture of lipid in this proteoglycan and suggest a location for the molecule in the plasma membrane. This proteoglycan was found in all cell species examined. - The other type of heparan sulfate proteoglycan had a larger molecular size than most previously described heparan sulfate proteoglycans and had a buoyant density around 1.32 g/ml, probably due to an unusually high ratio of protein to carbohydrate. This heparan sulfate proteoglycan was found only in extracts of cells capable of forming a fibrillar extracellular matrix, but not in extracts of cells devoid of matrix. It was retained in cell-free preparations of extracellular matrix, indicating that it may be a specific product of this compartment.     

Modification of proteoglycans during maturation of fibroblast substratum adhesion sites

Newly formed adhesion sites, left bound to the tissue culture substratum after [ethylenebis(oxyethylenenitrilo)] tetraacetic acid mediated detachment of simian virus 40 transformed Balb/c 3T3 cells, have been extracted with 0.5 M guanidine hydrochloride or Zwittergent (3-12), extractions which identify different subfractions of proteoglycans in these sites. The compositions of these extracts were then compared to similar extracts of "maturing" adhesion sites in an effort to identify structural and metabolic changes which may occur with time and which may play a role in altering adhesion during cell movement. Guanidine hydrochloride (0.5 M) extracts both hyaluronate and chondroitin sulfate proteoglycan from newly formed sites (but which are not complexed in an aggregate similar to that found in cartilage) but only hyaluronate from fully matured sites, indicating that the chondroitin sulfate proteoglycans somehow become resistant to extraction with time. Both high and low molecular weight forms of hyaluronate also accumulate in sites with time. Zwittergent 3-12 solubilizes free chains of heparan sulfate but not heparan sulfate proteoglycan from either class of sites. Most of the heparan sulfate in newly formed sites occurs as a large proteoglycan excludable from Sepharose CL-6B columns under stringent dissociative conditions; however, as adhesion sites "mature", a portion of this proteoglycan appears to be converted by some unknown mechanism to free heparan sulfate chains. This process may very well weaken the close adhesive contacts between the cell and substratum mediated by fibronectin's binding to the highly multivalent heparan sulfate proteoglycans. These studies further indicate that there is considerable metabolism and changing intermolecular associations of proteoglycans within these sites during movement of fibroblasts over this model extracellular matrix.     

Augmented synthesis and differential localization of heparan sulfate proteoglycans in Duchenne muscular dystrophy

Muscular dystrophies are characterized by continuous cycles of degeneration and regeneration that result in extensive fibrosis and a progressive diminution of muscle mass. Cell surface heparan sulfate proteoglycans are found almost ubiquitously on the surface and in the extracellular matrix (ECM) of mammalian cells. These macromolecules interact with a great variety of ligands, including ECM constituents, adhesion molecules, and growth factors. In this study, we evaluated the expression and localization of three heparan sulfate proteoglycans in the biopsies of Duchenne muscular dystrophy (DMD) patients. Through SDS-PAGE analyses followed by specific identification of heparitinase-digested proteins with an anti-Delta-heparan sulfate specific monoclonal antibodies, we observed an increase of three forms of heparan sulfate proteoglycans, corresponding to perlecan, syndecan-3, and glypican-1. Immunohistochemistry analyses indicated a differential localization for these proteoglycans: glypican-1 and perlecan were found mainly associated to ECM structures, while syndecan-3 was associated to muscle fibers. These results suggest that the amount of specific heparan sulfate proteoglycans is augmented in skeletal muscle in DMD patients presenting a differential localization.