Visualization of the large heparan sulfate proteoglycan from basement membrane

Kleinschmidt spreading, negative staining, and rotary shadowing were used to examine the large form of (basement membrane) heparan sulfate proteoglycan in the electron microscope. Heparan sulfate proteoglycan was visualized as consisting of two parts: the core protein and, emerging from one end of the core protein, the glycosaminoglycan side chains. The core protein usually appeared as an S-shaped rod with about six globules along its length. Similar characteristics were observed in preparations of core protein in which the side chains had been removed by heparitinase treatment ("400-kDa core") as well as in a 200-kDa trypsin fragment ("P200") derived from one end of the core protein. The core protein was sensitive to lyophilization and apparently also to the method of examination, being condensed following Kleinschmidt spreading (length means = 52 nm) and extended following negative staining (length means = 83 nm) or rotary shadowing (length means = 87 nm; 400-kDa core length means = 80 nm; P200 length means = 44 nm). Two or three glycosaminoglycan side chains (length means = 146 +/- 53 nm) were attached to one end of the core protein. The side chains often appeared tangled or to merge together as one. Thus, the large heparan sulfate proteoglycan from basement membrane is an asymmetrical molecule with a core protein containing globular domains and terminally attached side chains. This structure is in keeping with that previously predicted by enzymatic digestions and with the proposed orientation in basement membranes, i.e., the core protein bound in the lamina densa and the heparan sulfate side chains in the lamina lucida arranged along the surface of the basement membranes.     

Immunostaining of a heterodimeric dermatan sulphate proteoglycan is correlated with smooth muscles and some basement membranes

A heterodimeric 760-kDa dermatan sulphate proteoglycan tentatively named PG-760 was characterized as a product of keratinocytes, endothelial cells, and fibroblasts. The two core proteins of 460 kDa and 300 kDa are linked by disulphide bridges, and both carry one or only very few dermatan sulphate chains. Different antisera against PG-760 were used in the present study to investigate the distribution in selected murine tissues by light and electron microscopy. PG-760 immunostaining was observed in cornea (epithelium including basement membrane, stroma, and Descemet's membrane), skin, mucosa of the small intestine, Engelbreth-Holm-swarm (EHS)-tumour (matrix and cells), and the smooth muscle layers of uterus, small intestine, and blood vessels. No staining was observed in capillaries, striated muscles, and liver parenchyma including the central vein. The expression of PG-760 in EHS-tumour was also demonstrated after extraction with 4M guanidine and partial purification by diethylaminoethyl (DEAE)-chromatography. We conclude that this novel proteoglycan exhibits a unique tissue distribution being a constituent of some but not all basement membranes, of some other extracellular matrices, and additionally, of all investigated smooth muscle layers.     

Transformed mouse mammary epithelial cells synthesize undersulfated basement membrane proteoglycan

Proteoglycans deposited in the basal lamina of [14C] glucosamine-labeled normal and [3H]glucosamine-labeled transformed mouse mammary epithelial cells grown on type I-collagen gels, were extracted in 4 M guanidinium chloride and cofractionated over Sepharose CL 4B. The heparan sulfate chains carried by these proteoglycans were isolated by treatment with alkaline borohydride, protease K, chondroitinase ABC, and cetylpyridinium chloride precipitation. Heparan sulfate isolated from transformed cell cultures consistently eluted from DEAE-cellulose at lower salt concentrations and was of smaller apparent Mr when chromatographed over Sepharose CL 6B, than heparan sulfate of normal cell cultures. Experiments using doubly labeled cultures ([3H]glucosamine and [35S]sulfate) demonstrated an approximately 30% reduction in the sulfate/hexosamine ratio in heparan sulfate derived from transformed cultures. Both N- and O-sulfate were decreased. The decreased Mr and decreased sulfation of heparan sulfate upon transformation appear sufficient to explain the altered heparan sulfate/chondroitin sulfate ratios previously observed in these cells. These changes may have implications for the molecular interactions in which these proteoglycans are normally engaged during basal lamina assembly, and cause the poor basal lamina formation displayed by these transformed cells.     

Characterization of heparan sulfate proteoglycan from calf lens capsule and proteoglycans synthesized by cultured lens epithelial cells. Comparison with other basement membrane proteoglycans

After extraction with 4 M guanidinium chloride and purification by DEAE-cellulose chromatography, the heparan sulfate proteoglycan (HSPG) of calf anterior lens capsule was found to consist of two immunologically related components (Mr = 340,000 and 250,000) which upon deglycosylation with trifluoromethanesulfonic acid yielded core proteins with Mr values of 170,000 and 145,000. The heparan sulfate chains were uniform in size (Mr = 14,000) and manifested a clustering of sulfate groups in a peripheral domain. From the decrease in Mr observed after heparitinase digestion, it could be estimated that 6 and 11 glycosaminoglycan chains were present in the Mr = 250,000 and 340,000 components respectively. The occurrence of N-linked oligosaccharides was evident from the size difference of the heparitinase- and trifluoromethane-sulfonic acid-treated proteoglycans (approximately 20 kDa), as well as from the presence of a substantial number of mannose residues; furthermore, interaction of the capsule proteoglycan with Bandeiraea simplicifolia I suggested that these carbohydrate units contains terminal alpha-D-Gal groups. Cultured lens epithelial cells deposited a single [35S]sulfate-labeled proteoglycan into their matrix (Mr = 400,000) which was immunologically related to the lens capsule proteoglycan and contained only heparan sulfate chains. In addition to this component, the medium from these cells contained an immunologically unrelated HSPG (Mr = 150,000) as well as a chondroitin sulfate proteoglycan (Mr = 240,000). Examination of bovine glomeruli indicated that, in addition to the previously described 200-kDa HSPG, an immunologically related 350-kDa component was also present. This size heterogeneity, which is comparable to that seen in the lens capsule, is most readily attributable to proteolytic processing of a precursor molecule. Studies with polyclonal antibodies demonstrated only limited cross-reactivities between the Engelbreth-Holms-Swarm proteoglycan and the components from lens capsule and glomerular basement membrane; since even the latter two differed somewhat in their antigenic sites, it would appear that cell- and species-dictated genetic differences as well as post-translational events contribute to the diversity observed in basement membrane HSPGs.     

beta-D-xyloside-mediated alteration in the synthesis of basement membrane proteoglycan

The effect of nitrophenyl-beta-D-xyloside (xyloside), a synthetic initiator of glycosaminoglycan synthesis, on proteoglycan and glycosaminoglycan synthesis by a basement membrane producing tumor was studied. While xyloside markedly stimulated the formation of chondroitin sulfate chains, it depressed the formation of a basement membrane heparan sulfate proteoglycan and caused only little formation of free heparan sulfate chains. However, when the synthesis of the core protein of the proteoglycan was inhibited by cycloheximide, heparan sulfate chains were produced by xyloside treatment. These heparan sulfate chains had a sulfate content higher than that of heparan sulfate found on the proteoglycan. The data indicate that xyloside can substitute for the heparan sulfate initiation site on the core protein of the proteoglycan and that this initiation is enhanced in the absence of core protein. This suggests that under normal conditions the formation of heparan sulfate chains may be tightly linked to the production of the core protein.     

Heterogeneous distribution of a basement membrane heparan sulfate proteoglycan in rat tissues

A heparan sulfate proteoglycan (HSPG) synthesized by murine parietal yolk sac (PYS-2) cells has been characterized and purified from culture supernatants. A monospecific polyclonal antiserum was raised against it which showed activity against the HSPG core protein and basement membrane specificity in immunohistochemical studies on frozen tissue sections from many rat organs. However, there was no reactivity with some basement membranes, notably those of several smooth muscle types and cardiac muscle. In addition, it was found that pancreatic acinar basement membranes also lacked the HSPG type recognized by this antiserum. Those basement membranes that lacked the HSPG strongly stained with antisera against laminin and type IV collagen. The striking distribution pattern is possibly indicative of multiple species of basement membrane HSPGs of which one type is recognized by this antiserum. Further evidence for multiple HSPGs was derived from the finding that skeletal neuromuscular junction and liver epithelia also did not contain this type of HSPG, though previous reports have indicated the presence of HSPGs at these sites. The PYS-2 HSPG was shown to be antigenically related to the large, low buoyant density HSPG from the murine Engelbreth-Holm swarm tumor. It was, however, confirmed that only a single population of antibodies was present in the serum. Despite the presence of similar epitopes on these two proteoglycans of different hydrodynamic properties, it was apparent that the PYS-2 HSPG represents a basement membrane proteoglycan of distinct properties reflected in its restricted distribution in vivo.     

Heparan sulfate proteoglycan is present in basement membrane as a double-tracked structure

Basement membranes contain 4.5-nm wide sets of two parallel lines, along which short prongs called "spikes" occur at regular intervals. The nature of this structure, referred to as "double tracks," was investigated in Lowicryl sections of mouse kidney and rat Reichert's membrane immunolabeled for basement membrane components using secondary antibodies conjugated to 5-nm gold particles. When the mouse glomerular basement membrane and rat Reichert's membrane were exposed to antibodies directed to the core protein of heparan sulfate proteoglycan, 95% or more of the gold particles were over double tracks, whereas after exposure of Reichert's membrane to antisera against laminin, collagen IV, or entactin, labeling of the double tracks remained at the random level. When heparan sulfate proteoglycan was incubated in Tris buffer, pH 7.4, at 35 degrees C for 1 hr, a precipitate resulted which, on electron microscopic examination, was found to consist of 5- to 6-nm wide sets of two parallel lines along which densities were observed. Immunolabeling confirmed the presence of the proteoglycan's core protein in the sets. Since double tracks were closely similar to this structure and were labeled with the same antibodies, they were likely to be also composed of heparan sulfate proteoglycan.     

Domain structure of the basement membrane heparan sulfate proteoglycan

We have used proteolytic digestions and immunological reactivity to map regional domains of the 400-kilodalton (kDa) core protein of the heparan sulfate containing basement membrane proteoglycan from the Englebreth-Holm-Swarm tumor. Digestion with V8 protease caused the rapid release of numerous large peptides ranging in size from 80 to 200 kDa and a 44-kDa peptide. The 44-kDa peptide (P44) was stable to further digestion, but the larger peptides were eventually degraded to a 46-kDa peptide (P46). Both the P44 and P46 fragments migrate slower in the presence of a reducing agent, indicating intrachain disulfide bonding, and do not have heparan sulfate side chains. Antisera to the P46 fragment, however, did not react with P44 fragment, and the amino acid compositions of P46 and P44 fragments were different. This suggests that these two fragments were unrelated. Trypsin digestion of the proteoglycan immediately released a 200-kDa peptide (P200) that also lacked heparan sulfate side chains. Digestion of the P200 fragment with V8 protease produced the P44 and P46 fragments in the same temporal sequence seen with V8 protease digestion of the proteoglycan. Antisera to the P200 fragment reacted strongly with the P44 and P46 fragments. These results show that the P44 and P46 domains are contained within the P200 domain. The rapid release of the P44 domain indicates that it is located at one end of the core protein. The large size of these proteolytic fragments suggests the core protein contains considerable conformational structure, and the absence of heparan sulfate on the P200 domain indicates that the side chains are asymmetrically located on the core.     

Synthesis and effects of basement membrane components in cultured rat Schwann cells

Schwann cells, the myelin-forming cells of the peripheral nervous system, are surrounded by a basement membrane. Whether cultured rat Schwann cells synthesize the basement membrane-specific components, laminin and collagen type IV, and whether these components influence the adhesion, morphology, and growth of these cells have been investigated. Both laminin and collagen type IV were detected in the cytoplasm of Schwann cells by immunofluorescence. After ascorbate treatment, laminin and collagen type IV were both found in an extracellular fibrillar matrix bound to the Schwann cell surface. Laminin was further localized on the Schwann cell surface by electron microscopy using gold immunolabeling. Anti-laminin IgG-labeled gold particles were scattered over the cell surface, and linear rows of particles and small aggregates were found along the cell edges and at points of contact with other cells. When added to the culture medium, laminin acted as a potent adhesion factor, stimulating Schwann cell adhesion as much as eightfold above control levels on type IV collagen. In the presence of laminin, the cells became stellate and by 24 hr had extended long, thin processes. Laminin also stimulated cell growth in a dose-dependent manner and anti-laminin IgG completely inhibited cell attachment and growth in the absence of exogenous laminin. Thus, cultured Schwann cells synthesize laminin and collagen type IV, two major components of basement membrane, and laminin may trigger Schwann cell differentiation in vivo during early stages of axon-Schwann cell interaction before myelination.     

Lack of heparan sulfate proteoglycan in a discontinuous and irregular placental basement membrane

A discontinuous basement membrane of variable width that surrounds spongiotrophoblast cells of rat placenta was examined for the presence of type IV collagen, laminin, a heparan sulfate proteoglycan, entactin, and fibronectin using monospecific antibodies or antisera and the indirect peroxidase technique. At the level of the light microscope, the basement membrane was immunostained for type IV collagen, laminin, entactin, and fibronectin. Heparan sulfate proteoglycan immunostaining, however, was virtually absent even after pretreatment of sections with 0.1 N acetic acid, pepsin (0.1 microgram/ml) or 0.13 M sodium borohydride. Examination in the electron microscope confirmed the lack of immunostaining for heparan sulfate proteoglycan, whereas the other substances were mainly localized to the lamina densa part of the basement membrane. The absence of heparan sulfate proteoglycan in this discontinuous and irregular basement membrane even though type IV collagen, laminin, entactin, and fibronectin are present, suggests that heparan sulfate proteoglycan may have a structural role in the formation of basement membrane.     

High concentrations of low density lipoprotein decrease basement membrane-associated heparan sulfate proteoglycan in cultured endothelial cells

The effect of increasing low density lipoprotein (LDL) concentrations on the synthesis of basement membrane components was investigated in proliferating porcine aortic endothelial cells (PAEC) in culture. Basement membrane-associated heparan sulfate proteoglycan (HSPG) and fibronectin were determined by enzyme immunoassay. Low extracellular LDL-levels increase, high extracellular LDL-levels decrease the HSPG content of PAEC. Fibronectin synthesis was only slightly affected while proliferation and metabolic activity as assessed by lactate production were constant. Insulin or high extracellular glucose did not influence the effect of LDL on basement membrane components.     

Synthesis of basement membrane proteins by rat mammary epithelial cells

A mammary epithelial cell line, Rama 25, growing on plastic, deposits fibronectin, type IV collagen, and laminin in punctate structures located beneath the basal surface of the cells. When grown on the surface of collagen gels, Rama 25 cells deposit these basement membrane proteins in a continuous layer between the basal surface of the cells and the surface of the collagen matrix. Rama 25 cells also penetrate the collagen matrix forming rudimentary duct-like structures. These structures are surrounded by a discontinuous layer of basement membrane proteins. The ducts of fetal and neonatal rat mammary glands contain few mature myoepithelial cells and our results suggest that some mammary epithelial cells, in contact with a collagenous stroma, are capable of synthesizing a basal lamina-like structure.     

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.     

Reduced synthesis of basement membrane heparan sulfate proteoglycan in streptozotocin-induced diabetic mice

In diabetes, certain basement membranes become thicker yet more porous than normal. To identify possible changes in the basement membrane, we have grown the Engelbreth-Holm-Swarm tumor, a tissue that produces quantities of basement membrane in normal mice and in streptozotocin-treated, insulin-deficient, diabetic mice. The level of laminin, a basement membrane-specific glycoprotein, and the level of total protein were slightly elevated in the diabetic tissue. In contrast, the level of the basement membrane specific heparan sulfate proteoglycan was only 20% of control. The synthesis of this proteoglycan was also reduced in the diabetic animals, while the synthesis of other proteoglycans by tissues such as cartilage was normal. The synthesis of the heparan sulfate proteoglycan in diabetic animals was inversely related to plasma glucose levels showing an abrupt decrease above the normal range of plasma glucose. Insulin restored synthesis to normal but this required doses of insulin that maintained plasma glucose at normal levels for several hours. Since the heparan sulfate proteoglycan in the basement membrane restricts passage of proteins, its absence could account for the increased porosity of basement membrane in diabetes. A compensatory synthesis of other components could lead to their increased deposition and the accumulation of basement membrane in diabetes.     

Stimulation of large proteoglycan synthesis in cultured smooth muscle cells from pig aorta by endothelial cell-conditioned medium.

We have previously shown (Berrou et al., J. Cell. Phys., 137:430-438, 1988) that porcine endothelial cell-conditioned medium (ECCM) stimulates proteoglycan synthesis by smooth muscle cells from pig aorta. ECCM stimulation requires protein cores for glycosaminoglycan chain initiation and is accompanied by an increase in the hydrodynamic size of proteoglycans secreted into the medium. This work investigates the mechanisms involved in the ECCM effect. 1) Control and ECCM stimulated proteoglycan synthesis (measured by a 20 min [35S]-sulfate labeling assay) was not inhibited by cycloheximide, indicating that the proteoglycans were composed of preexisting protein cores and that ECCM stimulates glycosylation of these protein cores. 2) Whereas ECCM stimulation of [35S]-methionine incorporation into secreted proteins only occurred after a 6 h incubation, the increase in [35S] methionine-labeled proteoglycans was observed after 1 h, and the increase was stable for at least 16 h. 3) As analysed by electrophoresis in SDS, chondroitinase digestion generated from [14C] serine-labeled proteoglycans 7 protein cores of high apparent molecular mass (550-200 kDa) and one of 47 kDa. The two protein cores of highest apparent molecular masses (550 and 460 kDa), but not the 47 kDa protein cores, showed increased [14C]-serine incorporation in response to ECCM (51%, as measured by Sepharose CL-6B chromatography). 4) Finally, incorporation of [35S]-sulfate into chondroitinase-generated glycosaminoglycan linkage stubs on protein cores was determined by Sepharose CL-6B chromatography: ECCM did not modify the ratio [35S]/[14C] in stimulated protein cores, indicating that ECCM did not affect the number of glycosaminoglycan chains. The results of these studies reveal that 1) endothelial cells secrete factor(s) that preferentially stimulate synthesis of the largest smooth muscle cell proteoglycans without structural modifications and 2) the stimulation proceeds via increased glycosylation of protein core through enhancement of xylosylated protein core, followed by enhanced protein synthesis.     

High affinity interactions between the Alzheimer's beta-amyloid precursor proteins and the basement membrane form of heparan sulfate proteoglycan

High affinity interactions were studied between the basement membrane form of heparan sulfate proteoglycan (HSPG) and the 695-, 751-, and 770-amino acid Alzheimer amyloid precursor (AAP) proteins. Based on quantitative analyses of binding data, we identified single binding sites for the HSPG on AAP-695 (Kd = 9 x 10(-10) M), AAP-751 (Kd = 10 x 10(-9) M), and AAP-770 (Kd = 9 x 10(-9) M). It is postulated that the "Kunitz" protease inhibitor domain which is present in AAP-751 and -770 reduces the affinity of AAPs for the HSPG through steric hindrance and/or conformational alteration. HSPG binding was inhibited by heparin and dextran sulfate, but not by dermatan or chondroitin sulfate. HSPG protein core, obtained by heparitinase digestion, also bound to the beta-amyloid precursor proteins with high affinity, indicating that the high affinity binding site is constituted by the polypeptide chain rather than the carbohydrate moiety. The effects of various cations on these interactions were also studied. Our results suggest that specific interactions between the AAP proteins and the extracellular matrix may be involved in the nucleation stages of Alzheimer's disease type amyloidogenesis.     

Glomerular basement membrane proteoglycans are derived from a large precursor

The basement membrane heparan sulfate proteoglycan produced by the Englebreth-Holm-Swarm (EHS) tumor and by glomeruli were compared by immunological methods. Antibodies to the EHS proteoglycan immunoprecipitated a single precursor protein (Mr = 400,000) from [35S]methionine-pulsed glomeruli, the same size produced by EHS cells. These antibodies detected both heparan sulfate proteoglycans and glycoproteins in extracts of unlabeled glomeruli and glomerular basement membrane. The proteoglycans contained core proteins of varying size (Mr = 150,000 to 400,000) with a Mr = 250,000 species being predominant. The glycoproteins are fragments of the core protein which lack heparan sulfate side chains. Antibodies to glomerular basement membrane proteoglycan immunoprecipitated the precursor protein (Mr = 400,000) synthesized by EHS cells and also reacted with most of the proteolytic fragments of the EHS proteoglycan. This antibody did not, however, react with the P44 fragment, a peptide situated at one end of the EHS proteoglycan core protein. These data suggest that the glomerular basement membrane proteoglycan is synthesized from a large precursor protein which undergoes specific proteolytic processing.     

The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells

Laminin and fibronectin are glycoproteins that influence cell behavior and mediate cell/substratum adhesion. We have examined the interaction of these macromolecules with the serine protease plasminogen activator (PA) in two types of extracellular matrices; one produced by the murine Engelbreth-Holm-Swarm (EHS) tumor (Matrigel), and another by normal kidney epithelial cells in culture. Matrigel was found to contain significant quantities of tissue-type PA (tPA). Two of the major components of Matrigel, laminin and type IV collagen, were also examined. Tissue-type PA was associated with purified preparations of laminin; however, it was not found associated with type IV collagen. Normal kidney epithelial cells in culture secrete large amounts of urokinase (UK) and deposit a subepithelial matrix containing both laminin and fibronectin. These matrix macromolecules were isolated from the deposited matrix by immunoprecipitation, examined by zymography, and found to contain UK. The potential role of this interaction in the mechanisms of cell migration and matrix remodeling is discussed.