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.     

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.     

Immunological characterization of a basement membrane-specific chondroitin sulfate proteoglycan

Reichert's membrane, an extraembryonic membrane present in developing rodents, has been proposed as an in vivo model for the study of basement membranes. We have used this membrane as a source for isolation of basement membrane proteoglycans. Reichert's membranes were extracted in a guanidine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate buffer followed by cesium chloride density-gradient ultracentrifugation under dissociative conditions. The proteoglycans were subsequently purified from the two most dense fractions (greater than 1.3 g/ml) by ion-exchange chromatography. Mice were immunized with the proteoglycan preparation and four mAbs recognizing the core protein of a high-density, buoyant chondroitin sulfate proteoglycan were raised. Confirmation of antibody specificity was carried out by the preparation of affinity columns made from each of the mAbs. Chondroitin sulfate proteoglycans (CSPGs) were purified from both supernatant and tissue fractions of Reichert's membranes incubated in short-term organ culture in the presence of radiolabel. The resultant affinity-purified proteoglycan samples were examined by gel filtration, SDS-PAGE, and immunoblotting. This proteoglycan is of high molecular weight (Mr = 5-6 x 10(5)), with a core protein of Mr = approximately 1.5-1.6 x 10(5) and composed exclusively of chondroitin sulfate chains with an average Mr = 1.6-1.8 x 10(4). In addition, a CSPG was purified from adult rat kidney, whose core protein was also Mr = 1.6 x 10(5). The proteoglycan and its core protein were also recognized by all four mAbs. Indirect immunofluorescence of rat tissue sections stained with these antibodies reveal a widespread distribution of this proteoglycan, localized specifically to Reichert's membrane and nearly all basement membranes of rat tissues. In addition to heparan sulfate proteoglycans, it therefore appears that at least one CSPG is a widespread basement membrane component.     

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.     

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.     

Isolation, characterization and immunological determination of basement membrane-associated heparan sulfate proteoglycan

Basement membrane-associated heparan sulfate proteoglycan (HSPG) was extracted from isolated porcine glomerular basement membranes and purified by ion-exchange chromatography. The proteogycan was characterized by specific enzymatic digestions, by amino-acid analysis, by SDS-polyacrylamide gel electrophoresis and by density gradient centrifugation. Polyclonal antibodies were raised against the purified HSPG in rabbits. Antibodies were characterized by enzyme immunoassays, immunoprecipitation and immunohistological methods. They were shown to recognize specifically the core protein of HSPG from porcine, human and rat glomerular basement membrane but did not recognize HSPG from guinea pig or rabbit kidney. The affinity-purified antibodies did not cross-react with other basement membrane proteins like laminin, fibronectin or collagen type IV nor with chondroitin sulfate-rich or keratan sulfate-rich proteoglycans from human or bovine tissue. Using these antibodies an enzyme immunoassay was developed for determination of HSPG in the range of 1-100 ng/ml. Studies with cultured porcine endothelial cells showed that subendothelial basement membrane-associated HSPG may be determined with the enzyme immunoassay.     

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.     

Origin and deposition of basement membrane heparan sulfate proteoglycan in the developing intestine

The deposition of intestinal heparan sulfate proteoglycan (HSPG) at the epithelial-mesenchymal interface and its cellular source have been studied by immunocytochemistry at various developmental stages and in rat/chick interspecies hybrid intestines. Polyclonal heparan sulfate antibodies were produced by immunizing rabbits with HSPG purified from the Engelbreth-Holm-Swarm mouse tumor; these antibodies stained rat intestinal basement membranes. A monoclonal antibody (mAb 4C1) produced against lens capsule of 11-d-old chick embryo reacted with embryonic or adult chick basement membranes, but did not stain that of rat tissues. Immunoprecipitation experiments indicated that mAb 4C1 recognized the chicken basement membrane HSPG. Immunofluorescent staining with these antibodies allowed us to demonstrate that distribution of HSPG at the epithelial-mesenchymal interface varied with the stages of intestinal development, suggesting that remodeling of this proteoglycan is essential for regulating cell behavior during morphogenesis. The immunofluorescence pattern obtained with the two species-specific HSPG antibodies in rat/chick epithelial/mesenchymal hybrid intestines developed as grafts (into the coelomic cavity of chick embryos or under the kidney capsule of adult mice) led to the conclusion that HSPG molecules located in the basement membrane of the developing intestine were produced exclusively by the epithelial cells. These data emphasize the notion already gained from previous studies, in which type IV collagen has been shown to be produced by mesenchymal cells (Simon- Assmann, P., F. Bouziges, C. Arnold, K. Haffen, and M. Kedinger. 1988. Development (Camb.). 102:339-347), that epithelial-mesenchymal interactions play an important role in the formation of a complete basement membrane.     

Identification of cDNA clones encoding different domains of the basement membrane heparan sulfate proteoglycan

We have used antibodies to the basement membrane proteoglycan to screen lambda gt11 expression vector libraries and have isolated two cDNA clones, termed BPG 5 and BPG 7, which encode different portions of the core protein of the heparan sulfate basement membrane proteoglycan. These clones hybridize to a single mRNA species of approximately 12 kilobases. Amino acid sequences obtained on peptides derived from protease digests of the core protein were found in the deduced sequence, confirming the identity of these clones. BPG 5 spanned 1986 base pairs and has an open reading frame of 662 amino acids. The amino acid sequence deduced from BPG 5 contains two cysteine-rich domains and two internally homologous domains lacking cysteine. The cysteine-rich domains show homology to the cysteine-rich domains of the laminin chains. A globule-rod structure, similar to that of the short arms of the laminin chains, is proposed for this region of the proteoglycan. The other clone, BPG 7, is 2193 base pairs long and has an open reading frame of 731 amino acids. The deduced sequence contains eight internal repeats with 2 cysteine residues in each repeat. These repeats show homology to the neural-cell adhesion molecule N-CAM and the plasma alpha 1B-glycoprotein. Looping structures similar to these proteins and to other proteins of the immunoglobulin gene superfamily are proposed for this region of the proteoglycan. The sequence DSGEY was found four times in this domain and could be heparan sulfate attachment sites.     

Structure of low density heparan sulfate proteoglycan isolated from a mouse tumor basement membrane

A large heparan sulfate proteoglycan of low buoyant density (p = 1.32 to 1.40 g/cm3 in 6 M-guanidine.HCl) was extracted from a tumor basement membrane with denaturing solvents and purified by chromatography and CsCl gradient centrifugation. Chemical, immunological, physical and electron microscopical analyses have demonstrated a high degree of purity and have allowed us to propose a structural model for this proteoglycan. It is composed of an 80 nm long protein core formed from a single polypeptide chain (Mr about 500,000) with intrachain disulfide bonds. This core is folded into a row of six globular domains of variable size as shown by electron microscopy after rotary shadowing and negative staining. A multidomain structure was confirmed by protease digestion experiments that allowed the isolation of a single heparan sulfate-containing peptide segment representing less than 5% of the total mass of the protein core. Electron microscopy has visualized generally three heparan sulfate chains in each molecule close to each other at one pole of the protein core. The molecular mass and length (100 to 170 nm) of the heparan sulfate chains were found to vary consistently between different preparations. The mass per length ratio (350 nm-1) indicated an extended conformation for the heparan sulfate side-chains. These structural features are distinctly different from those of the high density proteoglycan, suggesting that both forms of basement membrane heparan sulfate proteoglycan are genetically distinct and not derived from a common precursor.     

Self-assembly of a high molecular weight basement membrane heparan sulfate proteoglycan into dimers and oligomers

A high molecular weight basement membrane heparan sulfate proteoglycan, isolated from murine Englebreth-Holm-Swarm tumor, is seen in platinum replicas as an elongated flexible core (Mr = 450,000) consisting of a series of tandem globular domains from which extend, at one end, two to three heparan sulfate chains (average Mr = 80,000 each). This macromolecule will self-assemble into dimers and lesser amounts of oligomers when incubated in neutral isotonic buffer. These molecular species can be separated by zonal velocity sedimentation and assembly is seen to be time- and concentration-dependent. In rotary-shadowed platinum replicas the binding region is found at or near the end of the core at the pole opposite the origin of the heparan sulfate chains. Dimers are double-length structures and oligomers are seen as stellate clusters: in both, the heparan sulfate chains appear peripherally oriented. While isolated cores self-assemble, isolated heparan sulfate chains do not bind intact proteoglycans. Furthermore, proteolytic removal of a non-heparan sulfate containing core moiety destroys the ability of the proteoglycan monomer to form larger species or bind intact proteoglycan, further supporting the binding topography determined morphologically. These negatively charged macromolecular complexes may be important contributors to basement membrane structure and function.     

Isolation and partial characterization of heparan sulphate proteoglycan from the human glomerular basement membrane.

Heparan sulphate proteoglycan was solubilized from human glomerular basement membranes by guanidine extraction and purified by ion-exchange chromatography and gel filtration. The yield of proteoglycan was approx. 2 mg/g of basement membrane. The glycoconjugate had an apparent molecular mass of 200-400 kDa and consisted of about 75% protein and 25% heparan sulphate. The amino acid composition was characterized by a high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulphonic acid yielded core proteins of 160 and 110 kDa (and minor bands of 90 and 60 kDa). Alkaline NaBH4 treatment of the proteoglycan released heparan sulphate chains with an average molecular mass of 18 kDa. HNO2 oxidation of these chains yielded oligosaccharides of about 5 kDa, whereas heparitinase digestion resulted in a more complete degradation. The data suggest a clustering of N-sulphate groups in the peripheral regions of the glycosaminoglycan chains. A polyclonal antiserum raised against the intact proteoglycan showed reactivity against the core protein. It stained all basement membranes in an intense linear fashion in immunohistochemical studies on frozen kidney sections from man and various mammalian species.     

Deletion of the basement membrane heparan sulfate proteoglycan type XVIII collagen causes hypertriglyceridemia in mice and humans

Background

Lipoprotein lipase (Lpl) acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism.

Methods and Findings

We examined mutant mice defective in collagen XVIII (Col18), a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia.

Conclusions

This is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.     

A cytoskeleton-associated plasma membrane heparan sulfate proteoglycan in Schwann cells

Schwann cells cocultured with sensory neurons in a serum-free medium accumulate a single species of radiolabeled heparan sulfate proteoglycan (HS-PG) during incubation in medium containing 35SO4. This HS-PG was poorly extracted from cultures by solutions containing 1% Triton X-100 in low salt buffer or by solutions containing 1 M KCl, 4 M urea plus dithiothreitol, 1 mM Tris-HCl, 5 mM EDTA, or 100 micrograms/ml of heparin. The HS-PG was efficiently extracted, however, by 1% Triton X-100 in the presence of 1 M KCl or by 1% deoxycholate. These treatments solubilize both cell membranes and the Schwann cell cytoskeleton. In intact cells the HS-PG was digested by trypsin, indicating it was at least partially exposed on the cell surface. When solubilized HS-PG was applied to a column of octyl-sepharose CL-4B, more than 90% was retained by the column, but was quantitatively eluted by a solution containing 1% Triton X-100. In addition, the solubilized HS-PG could be incorporated into artificial phospholipid vesicles. These results indicate the HS-PG is an integral plasma membrane protein. The inability of low ionic strength solutions containing Triton X-100 to solubilize the HS-PG suggested it was bound to an additional structure. To determine whether the HS-PG was associated with the cytoskeleton we isolated cytoskeletons by detergent lysis of cells and centrifugation. The major protein components of isolated cytoskeletons were spectrin (Mr 225,000), vimentin (Mr 58,000), and actin (Mr 45,000). When 35SO4-labeled cells were used to prepare cytoskeletons approximately 80% of the total HS-PG was recovered in the cytoskeleton fraction. These results suggest the HS-PG is an externally exposed integral plasma membrane protein that is anchored to the Schwann cell cytoskeleton.     

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.     

Collagen XVIII,a basement membrane heparan sulfate proteoglycan,interacts with L-selectin and monocyte chemoattractant protein-1

Leukocyte infiltration during inflammation is mediated by the sequential actions of adhesion molecules and chemokines. By using a rat ureteral obstruction model, we showed previously that L-selectin plays an important role in leukocyte infiltration into the kidney. Here we report the purification, identification, and characterization of an L-selectin-binding heparan sulfate proteoglycan (HSPG) expressed in the rat kidney. Partial amino acid sequencing and Western blotting analyses showed that the L-selectin-binding HSPG is collagen XVIII, a basement membrane HSPG. The binding of L-selectin to isolated collagen XVIII was specifically inhibited by an anti-L-selectin monoclonal antibody, EDTA, treatment of the collagen XVIII with heparitinase or heparin but not by chemically desulfated heparin. A cell binding assay showed that the L-selectin-collagen XVIII interaction mediates cell adhesion. Interestingly, collagen XVIII also interacted with a chemokine, monocyte chemoattractant protein-1, and presented it to a monocytic cell line, THP-1, which enhanced the alpha(4)beta(1) integrin-mediated binding of the THP-1 cells to vascular cell adhesion molecule-1. Thus, collagen XVIII may provide a link between selectin-mediated cell adhesion and chemokine-induced cellular activation and accelerate the progression of leukocyte infiltration in renal inflammation.     

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).     

Identification of the precursor protein to basement membrane heparan sulfate proteoglycans

The precursor protein of a basement membrane specific heparan sulfate proteoglycan has been identified as a 400,000 Mr polypeptide. Antibodies against large and small forms of this proteoglycan, isolated from a basement membrane (Engelbreth-Holm-Swarm, EHS) tumor, immunoprecipitated the same 400,000 protein from pulse-labeled EHS cells. The proteoglycan precursor protein was not recognized by antibodies against other basement membrane components or by antibodies to the cartilage proteoglycan. Furthermore, heparan sulfate proteoglycan purified from the EHS tumor blocked the immunoprecipitation of the precursor protein. Pulse-chase studies with [35S]methionine showed the precursor protein was converted to a proteoglycan. Pulse-chase studies with 35SO4 showed the large, low density proteoglycan appeared first and was degraded to a smaller, high density proteoglycan. We propose that the precursor protein is used after very little or no modification in the assembly of a large, low density heparan sulfate proteoglycan and that a portion of the population of these macromolecules are subsequently degraded to a smaller form.