Characterization of novel sequences containing 3-O-sulfated glucosamine in glomerular basement membrane heparan sulfate and localization of sulfated disaccharides to a peripheral domain.

Fragmentation of the heparan sulfate chains from bovine glomerular basement membrane (GBM) by hydrazine/nitrous acid treatment followed by NaB3H4-reduction yielded a mixture of six sulfated disaccharides containing D-glucuronic (GlcUA) or L-iduronic acid (IdUA) and terminating in 2,5-anhydro[3H]mannitol (AnManH2), in addition to the nonsulfated component GlcUA beta 1----4AnManH2. Among these products two novel disaccharide units were identified as IdUA alpha 1----4AnManH2(3-SO4) and IdUA(2-SO4)alpha 1----4AnManH2(3-SO4); these accounted for 22% of the total sulfated species indicating that there are 2-3 residues of 3-O-sulfated glucosamine/heparan sulfate chain. The disulfated disaccharide was shown through its release by direct nitrous acid treatment to be situated in a GlcNSO3-IdUA(2-SO4)-GlcNSO3(3-SO4) sequence which is distinct from that in which 3-O-sulfated glucosamine is located in the antithrombin-binding region of heparins. Analyses of heparan sulfate from lens capsule, a nonvascular basement membrane, indicated the absence of sequences containing 3-O-sulfated glucosamine, although otherwise the sulfated disaccharides produced by hydrazine/nitrous acid/Na-B3H4 treatment (GlcUA beta 1----4AnManH2(6-SO4), IdUA alpha 1----4AnManH2(6-SO4), IdUA(2-SO4)alpha 1----4AnManH2 and IdUA(2-SO4)alpha 1----4AnManH2(6-SO4] were the same as from GBM. Examination of the GBM heparan sulfate domains after nitrous acid treatment indicated that the O- as well as N-sulfate groups are clustered in an iduronic acid-rich 10-disaccharide peripheral segment, while the internal region (approximately 20 disaccharides) is composed primarily of repeating GlcUA beta 1----4GlcNAc units. The localization of chain diversity to the outer region may facilitate interactions of the heparan sulfate with other macromolecular components.

Integrins of normal human epidermis: differential expression, synthesis and molecular structure.

The expression of integrin cell surface receptors in normal skin and their synthesis and molecular structure in keratinocyte cultures was investigated. The reactivity of four different polypeptides of the integrin family (alpha 2-, alpha 3-, alpha 6- and beta 1-chains) was demonstrated in the basal cell layer of normal epidermis. Studies of labelled keratinocyte cell lines showed that the polypeptides were expressed as alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 4 integrins. Only the alpha 6 beta 4 integrin showed polarization towards the basement membrane attachment site of basal layer keratinocytes, and was preferentially expressed at microvillous projections. In contrast, alpha 2 beta 1 and alpha 3 beta 1 integrins were equally expressed throughout the basal cell plasma membrane.

Loss and rearrangement of glomerular basement membrane laminin during acute nephrotoxic nephritis in the rat.

Many earlier studies have shown that the intravenous injection into rats of sheep antibodies against rat glomerular basement membrane (GBM) induces a rapid influx of neutrophils and proteinuria (nephrotoxic nephritis or NTN). The GBM antigens recognized by nephrotoxic antibodies (NTAbs) have not been identified conclusively. Our experiments presented here, however, showed that NTAbs did not significantly reduce binding of anti-laminin IgGs to laminin-coated enzyme-linked immunosorbent assay (ELISA) plates or to the GBM in vivo, indicating little cross-reactivity between the NTAbs and laminin. To evaluate possible changes in GBM architecture during acute stages of NTN, the ultrastructural distribution of laminin was determined by postfixation, postembedding immunogold labeling, and compared between normal and nephritic rats. The density of immunoreactive GBM laminin was significantly reduced in rats with acute NTN. In addition, conjugates of anti-laminin IgG and horseradish peroxidase were intravenously injected into rats that then received injections of NTAbs. Anti-laminin peroxidase conjugates were also injected after administering NTAbs. In both cases, an overall decrease in anti-laminin peroxidase reaction product was observed as compared to normal controls. The densest labeling was seen in the lamina rara interna, especially in areas of endothelial cell detachment. Some immunoperoxidase reaction product was also bound to basal surfaces of detaching endothelial cells, demonstrating the removal of at least some laminin from the GBM. A decrease in GBM binding of intravenously injected anti-laminin IgG, both before and after injection of rats with NTAbs, was also confirmed by postembedding immunogold labeling. Furthermore, morphometry showed that the GBM was significantly wider in nephritic rats than in controls, indicating a redistribution of laminin over a greatly increased area. These immunoultrastructural findings show, therefore, that GBM architecture is altered in the early phase of NTN.

Nonneoplastic and nonhyperplastic thymus in myasthenia gravis. An immunohistochemical study with double immunoenzymatic labeling of basement membrane and cellular components.

Histologically normal thymus (type A) in patients with myasthenia gravis (MG) was immunohistochemically compared with hyperplastic MG thymus (type B) and normal non-MG thymus. In formalin-fixed, paraffin-embedded sections of ten type A, ten type B, and eight non-MG cases, the thymic epithelium and other cellular components were stained in conjunction with the basement membrane by a double immunoenzymatic method. This technique demonstrated a moderate architectural disturbance in type A thymus, with distended perivascular space (PVS), elongated medullary epithelium, and disrupted basement membrane. These changes were more prominent in type B thymus but were minimal to lacking in non-MG thymus. Compared with those in non-MG thymus, the myoid cells in MG thymuses of both types tended to cluster around the Hassall's corpuscles, with a slight decrease in number in type B but not in type A. B-lymphocytes were present in type B, type A, and non-MG thymuses in that order of abundance; the cells were confined to the medullary parenchyma in the non-MG group but were numerous both in the PVS and medulla in the MG groups. T-lymphocytes were increased in the expanded PVS of type A and B MG thymuses. The number of interdigitating reticulum cells was similar in the three groups, but the cellular distribution was more dispersed in MG thymuses of both types. These findings, although previously described in type B thymus, have not been well recognized in type A thymus. They support the view that a common abnormality (presumably chronic thymitis), differing in degree only, underlies MG thymuses regardless of the presence of follicular hyperplasia.

Glomerular basement membrane: evidence for collagenous domain of the alpha 3 and alpha 4 chains of collagen IV.

A collagenous component(s) of Mr = 60K was extracted from glomerular basement membrane with urea and was purified. Upon digestion, it yielded a collagenase-resistant fragment(s) of Mr = 23.5K. Both component and fragment showed immunochemical identity with the noncollagenous domains of the new alpha 3 & alpha 4 chains of collagen IV. The component is characterized by a collagenous domain of about 280 residues and a noncollagenous domain of about 250 residues. These findings further establish these new chains as distinct entities of collagen IV.

Determination of apical membrane polarity in mammary epithelial cell cultures: the role of cell-cell, cell-substratum, and membrane-cytoskeleton interactions.

The membrane glycoprotein, PAS-O, is a major differentiation antigen on mammary epithelial cells and is located exclusively in the apical domain of the plasma membrane. We have used 734B cultured human mammary carcinoma cells as a model system to study the role of tight junctions, cell-substratum contacts, and submembraneous cytoskeletal elements in restricting PAS-O to the apical membrane. Immunofluorescence and immunoelectronmicroscopy experiments demonstrated that while tight junctions demarcate PAS-O distribution in confluent cultures, apical polarity could be established at low culture densities when cells could not form tight junctions with neighboring cells. In such cultures the boundary between apical and basal domains was observed at the point of cell contact with the substratum. Immunocytochemical analysis of these cell-substratum contacts revealed the absence of a characteristic basement membrane containing laminin, collagen (IV), and heparan sulfate proteoglycan. However, serum-derived vitronectin was associated with the basal cell surface and the cells were shown to express the vitronectin receptor on their basolateral membranes. Additionally, treatment of cultures with antibodies against the vitronectin receptor caused cell detachment. We suggest, then, that interactions between vitronectin and its receptor, are responsible for establishment of membrane domains in the absence of tight junctions. The role of cytoskeletal elements in restricting PAS-O distribution was examined by treating cultures with cytochalasin D, colchicine, or acrylamide. Cytochalasin D led to a redistribution of PAS-O while colchicine and acrylamide did not. We hypothesize that PAS-O is restricted to the apical membrane by interactions with a microfilament network and that the cytoskeletal organization is dependent upon cell-cell and cell-substratum interactions.

Ultrastructural immunogold studies of heparan sulphate proteoglycan in normal human glomeruli and glomerulonephritis.

The distribution of heparan sulphate proteoglycans (HSPG) has been investigated in normal human glomeruli, membranous glomerulonephritis, mesangial IgA disease, and anti-glomerular basement membrane disease. HSPG was localized using anti-bovine HSPG antibody and 10 nm gold-labelled secondary antibody on paraformaldehyde-fixed, Lowicryl K4M resin-embedded kidneys. HSPG was present in all glomeruli and there was a zonation of its distribution in that it was predominantly on the epithelial aspect of the glomerular basement membrane (GBM) and mesangium with little in the central regions of the mesangial matrix. In the cases of immune complex glomerulonephritis, no HSPG was found in the electron-dense deposits. These findings contrast with our previous studies using the same technique in which type IV collagen and fibronectin were found predominantly on the endothelial aspect of the GBM.

Heparan sulfate proteoglycan from human tubular basement membrane. Comparison with this component from the glomerular basement membrane.

Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins.

Identification of basement membrane components in eosinophilic globules in a case of Spitz's nevus.

A case of Spitz's nevus with eosinophilic globules was examined using antibodies for several components of the basement membrane. Aggregated tumor cells revealed the same characteristics as normal nevocytic nevi, that is, they were surrounded by laminin and type-IV collagen, whereas type-VII collagen was absent. All of these components of basement membranes, including type-VII collagen, were also found in eosinophilic globules, which were densely stained by these antibodies. It is assumed that these eosinophilic globules are essentially composed of basement membrane components, which are probably synthesized by epidermal and possibly also by melanocytic tumor cells.

The use of inert dehydration and glycol methacrylate embedding for immunogold localization of glomerular basement membrane components.

A protocol is described for the preparation of human pathology specimens without fixation, in order to perform immunocytochemistry at an ultrastructural level. Using the technique in conjunction with immunogold labeling, the basal lamina components type IV collagen, laminin, and heparan sulfate have been demonstrated in glomerular capillary loops in stored frozen human renal tissue. Tissue was thawed and immediately dehydrated with the inert cryoprotectant ethanediol (inert dehydration) followed by embedding in low-acid glycol methacrylate polymerized using the accelerator n,n-dimethylaniline. Tissue processed in this way retained superior antigenic activity when compared with tissue reprocessed from wax blocks and embedded in low-acid glycol methacrylate. Inert dehydration is a technique useful for the localization of processing sensitive epitopes in routine fresh or frozen archival pathologic material. Furthermore, high probe densities can be achieved without recourse to etching or enzyme treatments.

Basement membrane alterations during development and regression of tubular cysts.

Tubular cysts consisting of dilatation of the collecting ducts at the level of the subcapsular zone of the kidney were induced in newborn rabbits by a single injection of methylprednisolone acetate. We describe here the structural and compositional modifications of the tubular basement membrane (BM) during the formation, growth, and regression of the tubular cysts. During development of the tubular cysts the cystic BM appeared thickened and multilayered, with numerous matrix vesicles. Alcian blue- (AB) and ruthenium red- (RR) positive material distributed differently along the BM of control and cystic tubuli. While the amount of RR-positive material appeared increased in the cystic BM, no differences in the intensity of the AB staining could be discerned between normal and cystic tubuli. Immunofluorescent staining for laminin and type IV collagen appeared to be slightly decreased in the cystic tubuli. However, the amount of fibronectin appeared clearly increased. These changes in the cystic BM appear at the beginning of the tubular dilatation and are not observed in other renal BM. We suggest that there is a causal relationship between the modifications of the BM and the development of the tubular cysts. Glucocorticoids appear to modify the synthesis and/or secretion of the BM components. An abnormal BM should modify the spatial and chemical signals encoded within the BM that, in turn, could lead to abnormal behavior of the tubular cells. This may result in a loss of the normal developmental constraints imposed upon the tubular epithelium, which then undergoes cystic dilatation. During the regression of the cysts, the abnormalities of the BM progressively disappear. The sharp increase in the number of interstitial cells, which show close relationships with the components of the BM, suggests that these cells may be involved in the removal of the cyst BM.

Immunolocalization of basement membrane molecules in the stroma of salivary gland pleomorphic adenoma.

In order to determine the participation of basement membrane molecules in formation of its characteristic stroma, 30 cases of salivary gland pleomorphic adenoma were examined by immunohistochemical staining for type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. The stroma was histopathologically classified into four types: hyaline, fibrous, myxoid, and chondroid. Immunohistochemically, type IV collagen and laminin were more intensively localized in hyaline, fibrous and chondroid types of stroma, whereas heparan sulfate proteoglycan was more prominent in myxoid areas. The results suggest that the stroma contains these basement membrane components, which are possibly biosynthesized by epithelial tumor cells, and that histological variety of the stroma depends on proportion of local contents of each basement membrane molecule.

Characteristics and in vivo occurrence of type VIII collagen.

Type VIII collagen was isolated from bovine Descemet's membranes by pepsin treatment and salt fractionation, as described by Kapoor et al. [(1986) Biochemistry 25, 3930-3937]. Contaminating type IV collagen was removed by ion-exchange chromatography. Purified type VIII collagen consisted of two different polypeptide chains and, compared to the fiber forming collagens, showed a higher thermal stability. Corresponding fractions isolated from pepsinized human Ewing's sarcoma and fetal calf aorta reacted immunologically with a protein of similar molecular mass. After extraction of Descemet's membranes with guanidine hydrochloride, a peptide of about 60 kDa was obtained. This seems to be the tissue form of type VIII collagen.

Ultrastructural distribution of collagen types I-VI in aging human retinal vessels.

Retinal vessels from freshly enucleated human eyes were classified into three stages of the hyalinisation process. The distribution of collagen types I-VI within the vessel walls was studied ultrastructurally by immunogold labelling combined with the tissue preparation techniques of cryoultramicrotomy and London resin white embedding. Collagen types I, III, IV, V, and VI were found in large vessels, types I, IV, and V plus small amounts of III and VI in small vessels, and types I, IV, and V in capillaries. Hyalinised vessel walls consisted mainly of types I, IV, and VI collagen.

Prognostic significance of laminin in adenocarcinoma of the lung.

The distribution of laminin in tumor-associated basement membrane was immunohistochemically investigated in 115 cases of adenocarcinoma of the lung. The distribution of laminin was classified into continuous and discontinuous staining patterns. The incidence of the discontinuous pattern was less in early-stage disease than that in advanced stages (P less than 0.01). In patients with stage I, the incidence of discontinuous patterns was greater in short-term survivors than in long-term survivors (P less than 0.05). By contrast, in patients with stage III, the discontinuous pattern of laminin was frequently seen in both long-term survivors and short-term survivors, with no difference between the two groups. These data suggest that the discontinuous pattern of laminin in tumor-associated basement membrane reflects the spread and dissemination of tumor, hence a close relationship to the prognosis.

Glomerular basement membrane. Identification of a fourth chain, alpha 4, of type IV collagen.

The noncollagenous domain hexamer of collagen IV from bovine glomerular basement membrane was further investigated to determine the types of collagen chain from which subunits M2*b and M3 are derived. M2*b was shown to be a shorter form, containing 9 fewer residues, of M2*a which was previously established as the noncollagenous domain of a third chain, alpha 3, of collagen IV (Saus, J., Wieslander, J., Langeveld, J.P.M., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). M3 was identified as the noncollagenous domain of a fourth chain, alpha 4, of type IV collagen, on the basis of additional sequence data together with previous findings. A comparison of the collagenous-noncollagenous junction regions of alpha 3(IV) and alpha 4(IV) chains with those of classical alpha 1(IV) and alpha 2(IV) chains reveals structural information which provides a potential strategy for molecular cloning of these novel chains. The results further reveal the complexity of electrophoresis patterns of the hexamer and potential ambiguities in using one-dimensional patterns to determine whether molecular defects of collagen IV occur in pathological processes affecting basement membranes.

Ultrastructural localization of extracellular matrix components in human retinal vessels and Bruch's membrane.

We have used the electron microscopic immunogold technique to localize precisely various extracellular matrix components in human retinal vessels and Bruch's membrane. Collagen types IV and V, laminin, and heparan sulfate proteoglycan core protein were localized in basement membranes of retinal capillaries. In addition to the capillary basement membrane components, collagen types I and III and fibronectin were found in the basement membranes of retinal arterioles and venules. The basement membranes of choriocapillaries and retinal pigment epithelial cells in Bruch's membrane also showed a similar distribution of the retinal capillary basement membrane components. Both the inner and outer collagenous layers of Bruch's membrane contained collagen types I and III along with fibronectin, whereas collagen type VI was mostly limited to the central elastic lamina. The precise localization and distribution of various extracellular matrix components in human retinal vessels and Bruch's membrane may be important for understanding their normal function as well as their alteration in disease.

Characterization of type IV collagen NC1 monomers and Goodpasture antigen in human renal basement membranes.

Monomeric subunits of the globular domain of type IV collagen from human renal basement membrane were isolated and characterized. The monomers, M24, M26, M28+, and M28 , which have been identified previously in human glomerular basement membrane, were characterized by amino acid analysis, amino-terminal sequencing, and electrophoretic mobility. The results indicate that M24 and M26 are derived from alpha 1(IV) and alpha 2(IV) collagen chains, respectively. Amino-terminal sequencing revealed that M28+, and M28 correspond to the globular domain of novel collagen chains. M28 has been characterized as the principal target antigen in Goodpasture's syndrome and antiglomerular basement membrane (GBM) nephritis, and both M28 species are absent from the GBM in Alport familial nephritis. The cationic charge of M28 appears to be a consequence of a relatively high concentration of basic amino acids when compared with other monomers. Previous studies of bovine GBM have demonstrated chains with amino-terminal sequence homology to M28+ and M28 .

Affinity cytochemical labeling of glomerular basement membrane anionic sites using specific biotinylation and colloidal gold probes.

To label specific anionic sites on glomerular capillary wall structures, biotin was covalently linked to sialic acid residues by sequential treatment with mild peroxidation and biotin hydrazide, while carboxyl groups were biotinylated by exposure to the combination of biotin hydrazide and a water-soluble carbodiimide reagent. Optimal specific labeling of rat glomerular structures was obtained with in situ perfusion of the biotinylation reagents, followed by fixation in 4% phosphate-buffered paraformaldehyde, embedment in LR White resin, and post-embedment detection of biotinylated sites using a sequence of anti-biotin antibodies followed by a secondary antibody-colloidal gold conjugate. Attempts to use streptavidin-gold conjugates were not successful. Specificity of labeling was confirmed by enzymatic (neuraminidase) pre-treatment or by modification of carboxyl groups, as evaluated by electron microscopy and by solid-phase assays of solubilized glomerular basement membrane (GBM) components. In two rats with heavy proteinuria induced by doxorubicin (Adriamycin), a marked reduction in sialic acid residues within the GBM and on the epithelial cell surfaces was found, suggesting that reduced charge density attributable to abnormal sialylation may be important in the pathogenesis of nephrotic proteinuria.

Ultrastructures of the epithelial basement membrane and the subepithelial capillaries in rabbit palatine tonsils.

The ultrastructure of both the epithelial basement membrane and the subepithelial capillaries in rabbit tonsils was investigated using light and transmission electron microscopy of sections, and scanning electron microscopy of alkali-water macerated tissues. The basement membrane of the crypt epithelium was seen to consist of the lamina lucida, lamina densa and lamina fibroreticularis. The lamina fibroreticularis is made up of both fine and thick collagen fibrils. The basement membrane possesses numerous pores (0.5-20 microns in diameter) through which free cells migrate. The basement membrane overlying the follicle protrudes hemispherically towards the crypt lumen, while that over the interfollicular area forms many papillary projections. The capillaries are surrounded by collagen fibrillar sheaths invariably located below the collagen fibrillar sheet of the epithelial basement membrane. The capillaries immediately below the crypt epithelium, including switch-back loops of capillaries in the papillae, are fenestrated sinusoids (20-40 microns in diameter). Plasma cells, lymphocytes and macrophages are numerously gathered around the capillaries. Possible functional relations between these free cells and the fenestrated sinusoids are discussed.