Identification of extracellular matrix ligands for the heparan sulfate proteoglycan agrin.

Agrin is a major brain heparan sulfate proteoglycan which is expressed in nearly all basal laminae and in early axonal pathways of the developing central nervous system. To further understand agrin's function during nervous system development, we have examined agrin's ability to interact with several heparin-binding extracellular matrix proteins. Our data show that agrin binds FGF-2 and thrombospondin by a heparan sulfate-dependent mechanism, merosin and laminin by both heparan sulfate-dependent and -independent mechanisms, and tenascin solely via agrin's protein core. Furthermore, agrin's heparan sulfate side chains encode a specificity in interactions with heparin-binding molecules since fibronectin and the cell adhesion molecule L1 do not bind agrin. Surface plasmon resonance studies (BIAcore) reveal a high affinity for agrin's interaction with FGF-2 and merosin (2.5 and 1.8 nM, respectively). Demonstrating a biological significance for these interactions, FGF-2, laminin, and tenascin copurify with immunopurified agrin and immunohistochemistry reveals a partial codistribution of agrin and its ECM ligands in the chick developing visual system. These studies and our previous studies, showing that merosin and NCAM also colocalize with agrin, provide evidence that agrin plays a crucial role in the function of the extracellular matrix and suggest a role for agrin in axon pathway development.     

Chondroitin sulfate proteoglycan in the extracellular matrix of the canine superior olivary nuclei

The perineuronal extracellular matrix of the canine superior olivary nuclei was examined by the histochemical method. The extracellular matrix was stained with Alcian blue (pH 1.0 and 2.5), high iron diamine and ruthenium red. The staining intensity of Alcian blue in the extracellular matrix was remarkably reduced after chondroitinase ABC digestion but not after that of heparitinase or hyaluronidase. These results indicate that the extracellular matrix consists of proteoglycans and contains the chondroitin sulfate proteoglycan.     

Chondroitin sulfate proteoglycan expression during neuronal development.

Proteoglycans of developing chick brain were distinguished on the basis of reactivity with four well characterized antibody reagents (S103L, to the CS-rich domain; HNK-1, to 6-sulfated glucuronic acid; 1-C-3, to the HABr region and 5-D-4, to KS chains). One chondroitin sulfate proteoglycan reacted exclusively with S103L and 1-C-3 and not with the other two antibodies, hence is designated the S103L reactive brain CSPG. The other proteoglycan reacted exclusively with HNK-1 and 5-D-4 and not with S103L and 1-C-3, hence it is designated the HNK-1 reactive brain CSPG. In addition to these immunological distinctions, the S103L and HNK-1 CSPGs exhibited significant biochemical differences at both the protein and carbohydrate levels. Most interestingly, both CSPGs were found in all regions of the brain, and were expressed in a developmentally regulated pattern. The S103L CSPG was not detectable prior to embryonic day 7, increased to a maximum at day 13-15 and declined by day 20 in most brain regions examined. In contrast, the HNK-1 CSPG was present as early as embryonic day 4 and remained constant through hatching. Neuronal cultures established from embryonic day 6 (E6) cerebral hemispheres represent an in vitro paradigm that mimics in vivo neuronal development and differentiation. In this culture system we found that the expression of the S103L and HNK-1 CSPG followed a pattern similar to that observed in developing brain and further, that neurons are probably the sole source of S103L CSPG in cerebral cortex during neuroembryogenesis.     

Estradiol-stimulated turnover of heparan sulfate proteoglycan in mouse uterine epithelium

Heparan sulfate proteoglycans (HSPGs) and dermatan sulfate/chondroitin sulfate proteoglycans may be extracted from the uterine epithelium of immature mice by a 1-min exposure of the luminal surface of excised uteri to 1% Nonidet P-40 detergent. In mice that are treated with estradiol there is a marked increase in free heparan sulfate glycosaminoglycan in the extract. (a) By Sepharose exclusion chromatography the [35S]sulfate-labeled major HSPG had a nominal Mr of 200-250 X 10(3), consisting of a core protein of about 80-90 X 10(3) Mr with about 8-10 heparan sulfate glycosaminoglycan chains (Mr = 13 X 10(3)). The HSPG had a lower bouyant density (less than 1.45 g/ml) than the dermatan sulfate/chondroitin sulfate proteoglycan and was heterogeneous, as was evident in the fact that HSPG attained equilibrium over a wide range of CsCl densities and also showed nonuniform interaction with octyl-Sepharose. (b) Virtually all of the major HSPG was removed when the epithelium was isolated by proteolysis, indicating a cell surface localization. A smaller, less prominent HSPG (nominal Mr = 80 X 10(3)) was synthesized during the first 2 h after isolation. (c) Label and chase experiments with and without chloroquine showed that virtually all of the free heparan sulfate glycosaminoglycan chains derived from endocytosis and lysosomal degradation of the plasma membrane-associated HSPG. We conclude that estradiol stimulates endocytosis of HSPG, predominantly from the basolateral epithelial surface and suggest that this HSPG turnover may reflect changes associated with blastocyst attachment and invasion of the endometrium.     

Characterization of a novel heparan sulfate proteoglycan found in the extracellular matrix of liver sinusoids and basement membranes

A novel heparan sulfate proteoglycan (HSPG) present in the extracellular matrix of rat liver has been partially characterized. Proteoglycans were purified from a high salt extract of total microsomes from rat liver and found to consist predominantly (approximately 90%) of HSPG. A polyclonal antiserum raised against this fraction specifically recognized HSPG by immunoprecipitation and immunoblotting. The intact, fully glycosylated HSPG migrated as a broad smear (150-300 kD) by SDS-PAGE, but after deglycosylation with trifluoromethanesulfonic acid only a single approximately 40-kD band was seen. By immunocytochemistry this HSPG was localized in the perisinusoidal space of Disse associated with irregular clumps of basement membrane-like extracellular matrix material, some of which was closely associated with the hepatocyte sinusoidal cell surface. It was also localized in biosynthetic compartments (rough ER and Golgi cisternae) of hepatocytes, suggesting that this HSPG is synthesized and deposited in the space of Disse by the hepatocyte. The anti-liver HSPG IgG also stained basement membranes of hepatic blood vessels and bile ducts as well as those of kidney and several other organs (heart, pancreas, and intestine). An antibody that recognizes the basement membrane HSPG found in the rat glomerular basement membrane did not precipitate the 150-300-kD rat liver HSPG. We conclude that the liver sinusoidal space of Disse contains a novel population of HSPG that differs in its overall size, its distribution and in the size of its core protein from other HSPG (i.e., membrane-intercalated HSPG) previously described in rat liver. It also differs in its core protein size from HSPG purified from other extracellular matrix sources. This population of HSPG appears to be a member of the basement membrane HSPG family.     

Immunocytochemical localization of heparan sulphate proteoglycan in the rat submandibular gland

Summary Heparan sulphate proteoglycan is the predominant proteoglycan synthesized by the parenchymal cells of the rat submandibular gland. A polyclonal antibody was used to localize this proteoglycan in the adult rat submandibular gland. Localization was accomplished by indirect immunoperoxidase cytochemistry at the light and electron microscopic levels. Heparan sulphate proteoglycan was localized in a continuous, linear pattern in the lamina densa of the basement membrane surrounding all of the epithelial components of the gland as well as the basement membrane of the capillaries and small arterioles in the glandular stroma. In addition, heparan sulphate proteoglycan was seen in vesicles and pits along the acinar cell basal plasmalemma adjacent to the basement membrane and in the endoplasmic reticulum and Golgi apparatus of the acinar cells.     

Differential expression of insulin-like growth factors in the uterine epithelium and extracellular matrix during early pregnancy

We have studied the simultaneous expression of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) in the uterine epithelium and extracellular matrix during the time of trophoblast attachment and implantation. These studies reveal that IGF-I and IGF-II display different spatial and temporal patterns of expression during early pregnancy, and suggest a role for them in the process of attachment and implantation. Specifically, IGF-I is strongly expressed in the basal lamina which is the site of trophoblast invasion into the maternal stroma, and also in the apical epithelium, the site of initial trophoblast attachment. IGF-II is expressed to a lesser extent in the basal lamina, lateral plasma membranes and apical epithelium on day 3 but is only prominent apically at the time of implantation, suggesting a role in attachment.     

Isolation of the heparan sulfate proteoglycans from the extracellular matrix of rat skeletal muscle

We have previously shown that asymmetric collagen-tailed acetylcholinesterase (AChE) is anchored to the extracellular matrix (ECM) by heparan sulfate proteoglycans (HSPGs). Here we present our studies on the characterization of such PGs from the ECM of rat skeletal muscles. After radiolabeling with 35SO4 for 24h, PGs were extracted from the muscle ECM with 4.0 M guanidine-HCl containing protease inhibitors. PGs were subsequently isolated using sequential DEAE-Sephacel chromatography, digestion with chondroitinase ABC, and Sepharose CL-4B. Two different hydrodynamic size species of HSPGs were found. One type had a Mr of 4-6 X 10(5) (Kav = 0.25) as estimated by gel chromatography in the presence of 1% SDS and accounted for 75% of the total HSPGs. The other HSPG had a Mr 1.5-2.5 X 10(5) (Kav = 0.41). The glycosaminoglycan (GAG) side chains (Mr 20,000 and 12,000) were found composed only of heparan sulfate as determined by nitrous acid oxidation and heparitinase treatment. The large-sized HSPG, which is concentrated in synaptic regions, contains only GAG chains of Mr 20,000, suggesting that each HSPG contains only one kind of heparan sulfate chain in its structure. Our results definitively establish by biochemical criteria that the basement membrane of mammalian skeletal muscle contains HSPGs, the likely matrix receptor for the immobilization of the asymmetric collagen-tailed AChE at the neuromuscular junction.     

Hepatic heparan sulphate proteoglycan and the recycling of transferrin.

Heparan sulphate proteoglycan, labelled with [35S]sulphate, was prepared from rat livers for studies of its interaction with purified rat transferrin. Affinity chromatography of the preparation on columns of immobilized differic transferrin and apotransferrin showed that the proteoglycan possessed affinity for both types of matrices at pH 7.3 and that this affinity significantly increased at pH 5.6. The glycosaminoglycan chains liberated from the proteoglycan by heparan sulphate lyase also bound to apotransferrin, albeit less strongly, whereas the deglycosylated core protein exhibited virtually no interaction with this matrix. In the presence of the proteoglycan at pH 5.6, the release of iron from the N-lobe of transferrin was accelerated. These observations suggest that heparan sulphate proteoglycan from the liver can mimick some of the known functions of bona fide transferrin receptors and, hence, interaction with the proteoglycan may provide an alternative nondegradative pathway for transferrin through hepatic cells.     

Presence of unsulfated heparan chains on the heparan sulfate proteoglycan of human colon carcinoma cells. Implications for heparan sulfate proteoglycan biosynthesis

We provide direct evidence for the presence of unsulfated, but fully elongated heparan glycosaminoglycans covalently linked to the protein core of a heparan sulfate proteoglycan synthesized by human colon carcinoma cells. Chemical and enzymatic studies revealed that a significant proportion of these chains contained glucuronic acid and N-acetylated glucosamine moieties, consistent with N-acetylheparosan, an established precursor of heparin and heparan sulfate. The presence of unsulfated chains was not dependent upon the exogenous supply of sulfate since their synthesis, structure, or relative amount did not vary with low exogenous sulfate concentrations. Culture in sulfate-free medium also failed to generate undersulfated heparan sulfate-proteoglycan, but revealed an endogenous source of sulfate which was primarily derived from the catabolism of the sulfur-containing amino acids methionine and cysteine. Furthermore, the presence of unsulfated chains was not due to a defect in the sulfation process because pulse-chase experiments showed that they could be converted into the fully sulfated chains. However, their formation was inhibited by limiting the endogenous supply of hexosamine. The results also indicated the coexistence of the unsulfated and sulfated chains on the same protein core and further suggested that the sulfation of heparan sulfate may occur as an all or nothing phenomenon. Taken together, the results support the current biosynthetic model developed for the heparin proteoglycan in which unsulfated glycosaminoglycans are first elongated on the protein core, and subsequently modified and sulfated. These data provide the first evidence for the presence of such an unsulfated precursor in an intact cellular system.     

Chondroitin sulphate and heparan sulphate sulphation motifs and their proteoglycans are involved in articular cartilage formation during human foetal knee joint development

Novel sulphation motifs within the glycosaminoglycan chain structure of chondroitin sulphate (CS) containing proteoglycans (PGs) are associated with sites of growth, differentiation and repair in many biological systems and there is compelling evidence that they function as molecular recognition sites that are involved in the binding, sequestration or presentation of soluble signalling molecules (e.g. morphogens, growth factors and cytokines). Here, using monoclonal antibodies 3B3(-), 4C3 and 7D4, we examine the distribution of native CS sulphation motifs within the developing connective tissues of the human foetal knee joint, both during and after joint cavitation. We show that the CS motifs have broad, overlapping distributions within the differentiating connective tissues before the joint has fully cavitated; however, after cavitation, they all localise very specifically to the presumptive articular cartilage tissue. Comparisons with the labelling patterns of heparan sulphate (HS), HS-PGs (perlecan, syndecan-4 and glypican-6) and FGF-2, molecules with known signalling roles in development, indicate that these also become localised to the future articular cartilage tissue after joint cavitation. Furthermore, they display interesting, overlapping distributions with the CS motifs, reflective of early tissue zonation. The overlapping expression patterns of these molecules at this site suggests they are involved, or co-participate, in early morphogenetic events underlying articular cartilage formation; thus having potential clinical relevance to mechanisms involved in its repair/regeneration. We propose that these CS sulphation motifs are involved in modulating the signalling gradients responsible for the cellular behaviours (proliferation, differentiation, matrix turnover) that shape the zonal tissue architecture present in mature articular cartilage.     

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.     

Chondroitin sulfate proteoglycan form of the Alzheimer's beta-amyloid precursor.

The Alzheimer's amyloid beta protein is derived from a family of membrane glycoproteins termed amyloid precursor proteins (APP). Here we show that APP exists as the core protein of a chondroitin sulfate (CS) proteoglycan, ranging in apparent molecular size from 140 to 250 kDa, secreted by glial cell line C6. After partial purification on ion-exchange and gel chromatography, the secreted APP proteoglycan was recognized on Western blots by several antibodies specific to different regions of APP. Chondroitinase AC or ABC treatment of our samples completely eliminated the high molecular weight proteoglycan with a concomitant increase in the APP protein. This digested product reacted with an anti-stub antibody which recognizes 4-sulfated disaccharide. Sequencing of the N terminus of the core protein of this CS proteoglycan yielded 18 residues identical to the N terminus sequence of the mature APP. Quantitative analysis showed that, in this cell line, about 90% of the secreted nexin II form of APP occurs in the proteoglycan form, suggesting that the CS chains have a role in the biological function of this protein. The close proximity of two consensus CS attachment sites to both the N terminus of the amyloid beta protein and the secretase cleavage site, suggests that the CS chains may affect the proteolysis of APP and production of the amyloid beta protein.     

Association of heparan sulfate proteoglycan and laminin with the cytoskeleton in rat liver

Rats were injected with 35SO4 and after 2 h their livers were removed and used to prepare a detergent-insoluble cytoskeleton fraction. Spectrin, cytokeratins, and actin were major protein components of the isolated cytoskeletons. The cytoskeleton fraction accounted for approximately 14% of the total trichloroacetic acid-insoluble 35SO4 radioactivity incorporated into the liver. The cytoskeleton-associated radioactivity was present in a single species of macromolecule. This molecule was not present to a significant extent in the detergent-soluble fraction containing the cell supernatant and dissolved membrane proteins. Further characterization revealed the cytoskeleton-associated molecule was a heparan sulfate proteoglycan: it was eluted from a Sepharose CL-4B column under denaturing conditions at Kav = 0.4; following mild alkaline hydrolysis the radioactivity was eluted at a Kav = 0.7; when this material was subjected to nitrous acid hydrolysis all of the radioactivity was eluted near the column included volume. The isolated cytoskeletons contained attached nuclei. Pure nuclei isolated without associated cytoskeletal elements contained less than 1% of the total liver trichloroacetic acid-insoluble 35SO4 radioactivity and no detectable heparan sulfate proteoglycan. These results suggested that other matrix proteins might be associated with the liver cytoskeleton. When the subcellular distribution of laminin was monitored by immunostaining proteins transferred to nitrocellulose, laminin was detected exclusively in the cytoskeleton fraction. These results provide evidence for an association between extracellular connective tissue proteins and intracellular structural proteins.     

Isolation and partial characterization of a rat hepatoma heparan sulfate proteoglycan

A proteoglycan was isolated from a Morris rat hepatoma by sequential precipitations with ammonium sulfate and cetyl pyridinium chloride followed by chromatography on Sepharose CL-4B and DEAE-cellulose. The proteoglycan has a molecular weight of about 1.5 × 10⁵ with 40,000 molecular weight glycosaminoglycan side chains, identified as heparan sulfate based on resistance to chondroitinase and susceptibility to nitrous acid treatment. Immunological studies showed that the protein core of this proteoglycan is immunologically distinct from a rat yolk sac tumor chondroitin sulfate proteoglycan (Å. Oldberg, E. G. Hayman, and E. Ruoslahti, 1981,J. Biol. Chem.256, 10847–10852), but resembles a heparan sulfate proteoglycan isolated from a basement membrane-producing mouse tumor (J. R. Hassell, P.M. Robey, H.-J. Barrach, J. Wilczek, S. R. Rennard, and G. R. Martin, 1980, Proc. Nat. Acad. Sci. USA77, 4494–4498).     

The binding of heparin to the extracellular matrix of endothelial cells up-regulates the synthesis of an antithrombotic heparan sulfate proteoglycan

Exposure of endothelial cells to heparin and other antithrombotic drugs specifically stimulates the synthesis of an antithrombotic heparan sulfate (HS). In the present work, biotinylated heparin (BiotHep) was used to characterize the binding site(s) of heparin responsible for the stimulus in HS synthesis on endothelial cells. No differences were observed between biotinylated and non-biotinylated heparin in their ability to increase the synthesis of HS. In kinetic studies the BiotHep showed fast, saturable and specific binding with an apparent K(D) of 83 nM to adherent cells and 44 nM to the extracellular matrix (ECM) in the absence of cells. By confocal and electron microscopy, BiotHep bound only to the ECM, co-localizing with fibronectin. The same pattern of binding to the ECM was observed using heparin conjugated with FITC or Alexa Fluor 488 in the presence or absence of fetal calf serum. However, after degradation of HS, heparin binds to the cell surface, indicating that endogenous HS possibly occupied the heparin binding sites. Analyses by flow cytometry and confocal microscopy of cells with non-associated ECM, showed labeling of the cell surface using syndecan-4 monoclonal antibody as well as wheat germ agglutinin, but no binding of heparin. Furthermore, the stimulation in HS synthesis is not elicited by heparin in the absence of ECM. These results indicate that the stimulus for the synthesis of HS does not require binding of the heparin to the cell surface, and the signaling may be mediated through the ECM.     

Co-solubilization of asymmetric acetylcholinesterase and dermatan sulfate proteoglycan from the extracellular matrix of rat skeletal muscles

We have previously communicated that heparin released asymmetric acetylcholinesterase (AChE) from cholinergic synapses. Here we report studies showing that heparin, besides releasing asymmetric AChE from the skeletal muscle extracellular matrix (ECM), specifically solubilizes a dermatan sulfate proteoglycan (DSPG) which accounts for more than 95% of the 35S-released material. The co-solubilization of AChE and the proteoglycan opens up the possibility that both macromolecules could be involved in the formation of the soluble AChE complex observed after incubation of muscle homogenate with heparin. Our results suggest a possible association between asymmetric AChE and DSPG at the muscle ECM, moreover this work is the first report of the existence of DSPG at the skeletal muscle cell surface.