Collagen types XII and XIV are present in basement membrane zones during human embryonic development

The collagens constitute a large group of proteins in the extracellular matrix that can be divided into several distinct families. Collagen types XII and XIV belong to a subgroup of non-fibrillar-collagens termed (fibril-associated collagens with interrupted triple-helices) (FACIT) and may be involved in basement membrane regulation providing specific molecular bridges between fibrils and other matrix components. However, the tissue distribution of the two proteins during human embryogenesis is still unclear. As a first step toward the elucidation of their possible cell biological functions, we compared the distribution of the two collagens during human organogenesis at the light microscopical level. We detected specific differences between the expression patterns of the two molecules, which may be related to their respective function within the basement membrane zones during human embryonic development. For example, in the developing intestine, collagen type-XII was present in the basement membrane zones of epithelia and endothelia. However, collagen type-XIV was restricted to the mesothelial basement membrane zones. We conclude that both collagens might well be able to serve different functions during human embryonic development although their structures are highly similar.     

Molecular and biochemical aspects of nematode collagens.

Collagens are major structural proteins of nematode cuticles and basement membranes (basal laminae). The collagen proteins that form these structures differ in their biochemical and physical properties and are encoded by distinct gene families. Nematode basement membrane collagens are large proteins that show strong homology to basement membrane collagens of vertebrates. There appear to be 2 nonidentical basement membrane collagen genes in nematodes. Cuticle collagens are about one-sixth the size of basement membrane collagens and are encoded by a large family of 20-150 nonidentical genes. Cuticle collagens can be subdivided into 4 families based upon certain structural features in the proteins. The mature, extracellular forms of both types of collagen proteins are extensively cross-linked by disulfide bonds and are largely insoluble in the absence of a thiol-reducing agent. Cuticle collagens also are cross-linked by nonreducible covalent bonds that involve tyrosine residues. The experimental studies that have led to our current understanding of the structures of basement membrane and cuticle collagens are reviewed. Some previous questions about the physical properties of these proteins are reexamined in light of the primary sequence information now available for the proteins.     

The localization and synthesis of some collagen types in developing mouse embryos.

The location of type IV (basement membrane)collagen in early post-implantation mouse embryos was examined by immunoperoxidase reactions using a specific immunoglobulin raised against mouse lens capsule collagen. Reaction was positive in the earliest embryos studied--on the fifth day of gestation (the day of detection of the copulation plug is the first day). It was found only in the primitive endoderm adjacent to the blastocoelic cavity. Subsequently in development, strong staining reactions were found in the parietal endoderm, Reichert's membrane and an acellular layer which separates the visceral endoderm of the egg cylinder from the ectoderm. In tenth to eighteenth day visceral yolk sacs, the mesodermal portion was stained, which is consistent with the presence of basement membranes around blood vessels. The endodermal portion of the visceral yolk sac did not react, while small amounts were found in the amnion. By incubation of various embryonic tissues with tritiated amino acids, purification of the biosynthesized secreted collagens and their partial characterization, the differential expression of several collagen genes was detected. Identification of collagen types was made by: reaction with specific antibodies to type I and IV collagens; electrophoretic mobility; sensitivity to reduction and to collagenase; analysis of the proportions of 3-hydroxyproline, 4-hydroxyproline and hydroxylysine; and CNBr peptides. In agreement with the data of Minor et al. (1976a) for the rat, mouse parietal endoderm synthesizes large amounts of type IV collagen. In contrast to their findings, however, the 165,000 molecular weight polypeptide is not converted to one of 100,000 after reduction, alkylation and repepsinization (Dehm and Kefalides, 1978). The endoderm of the visceral yolk sac was shown to be synthesizing primarily type I collagen, while the mesoderm layer of this membrane synthesized both type I and IV collagens. Little or no type IV collagen synthesis was detected in the endoderm of the visceral yolk sac. If it is correct that the visceral endoderm of the early embryo makes a major contribution to the formation of the endoderm portion of the visceral yolk sac, then it is clear that a switch in collagen gene expression must occur as it does so.     

The potential role of human kidney cortex cysteine proteinases in glomerular basement membrane degradation

(1) The degradation of glomerular basement membrane and some of its constituent macromolecules by human kidney lysosomal cysteine proteinases has been investigated. Three cysteine proteinases were extracted from human renal cortex and purified to apparent homogeneity. These proteinases were identified as cathepsins B, H and L principally by their specific activities towards Z-Arg-Arg-NHMec, Leu-NNap and Z-Phe-Arg-NHMec, respectively, and their Mr on SDS-polyacrylamide gel electrophoresis under reducing conditions. (2) Cathepsins B and L, at acid pH, readily hydrolysed azocasein and degraded both soluble and basement membrane type IV and V collagen, laminin and proteoglycans. Their action on the collagens was temperature-dependent, suggesting that they are only active towards denatured collagen. Cathepsin L was more active in degrading basement membrane collagens than was cathepsin B but qualitatively the action of both proteinases were similar, i.e., at below 32 degrees C the release of an Mr 400,000 hydroxyproline product which at 37 degrees C was readily hydrolysed to small peptides. (3) In contrast, cathepsin H had no action on soluble or insoluble collagens or laminin but did, however, hydrolyse the protein core of 35S-labelled glomerular heparan sulphate-rich proteoglycan. (4) Thus renal cysteine proteinases form a family of enzymes which together are capable of degrading the major macromolecules of the glomerular extracellular matrix.     

Fibronectin-independent attachment of human gingival fibroblasts to interstitial and basement membrane collagens

We have studied the ability of human gingival fibroblasts (HGF) to attach to different interstitial (types I, II and III) and basement membrane (types IV and V) collagens. HGF cells were plated onto collagen-coated Petri dishes under various conditions and the percentage of cells attaching to the collagen was determined. HGF were found to attach to all the different types of native collagens, but attached poorly to the corresponding denatured collagens. When plated in the presence of 15% fetal bovine serum (FBS) or fibronectin-depleted FBS, similar percentages (approximately 85%) of cells attached to both interstitial and basement membrane collagens, demonstrating an attachment mechanism that is independent of plasma fibronectin. That the attachment in the presence of serum was also independent of cellular fibronectin was shown by the inability of fibronectin antibodies to block attachment to any of the collagen types. HGF were also capable of attaching to all of the collagen types in the complete absence of serum. In previous studies, investigators using cell lines have suggested that cell attachment in the absence of serum is non-physiological. However, the serum-free attachment of HGF to collagen was found to be dependent on cellular protein synthesis indicating that this attachment mechanism has biological significance.     

Sequential behaviour of extracellular matrix glycoproteins in an experimental model of hepatic fibrosis

The behaviour of extracellular matrix glycoproteins (fibronectin, laminin, basement membrane heparan-sulphate proteoglycan, type III, IV and V collagens) has been investigated in a sequential model of experimental hepatic fibrosis, using an immunofluorescence technique. The presence of some basement membrane macromolecules (such as type IV and V collagens, laminin and basement membrane heparan-sulphate proteoglycan) is detectable only in the early stages of septa formation, while type III collagen and fibronectin persist in late septa. These data suggest that hepatic fibroplasia proceeds through different steps in which stromal glycoproteins are preferentially engaged, as happens during organogenesis.     

Isolation of three collagenous components of probable basement membrane origin from several tissues

Fractionation of pepsin-solubilized collagens from several human tissues has shown that substantial quantities of collagen-like protein remain in solution under conditions leading to the precipitation of Type I, II, and III collagens. Characterization of the more soluble collagens has led to the isolation of three unique collagenous components each of which exhibit compositional features indicative of their origin from basement membranes. One of these has an apparent molecular weight of 55,000 daltons and appears to originate in endothelial basement membranes. The other two components (A chain and B chain) are somewhat larger than collagen α chains and appear to be derived from the collagen of epithelial and smooth muscle basement membranes, respectively.     

A developmental biologist’s “outside-the-cell” thinking

A major gap in our understanding of cell biology is how cells generate and interact with their surrounding extracellular matrix. Studying this problem during development has been particularly fruitful. Recent work on the basement membrane in developmental systems is transforming our view of this matrix from one of a static support structure to that of a dynamic scaffold that is regularly remodeled to actively shape tissues and direct cell behaviors.Cell biology is an enormously broad discipline that examines cell structure and function, as well as interactions between the cell and its environment. Studying cell biology during development offers one of the most dynamic, process-rich, and physiologically relevant settings for understanding the functions of cells. Thus, many seminal findings on cell signaling, the cell cycle, cell migration, cell polarization, and programmed cell death have been discovered in developmental contexts.One component of a cell’s environment, the extracellular matrix, is both of the cell and outside the cell, and its relationship with cells is therefore complex. In vitro studies have suggested roles for extracellular matrix in directing cell shape, differentiation, survival, and migration (Hay, 1981; Bernfield and Banerjee, 1982; Hadley et al., 1985; Goodman et al., 1989). Specific functions for extracellular matrix have been difficult to establish in vivo, however, because of the challenge of examining cell–matrix interactions in animals. Many vertebrate tissues encased with matrix are situated deep inside the organism and are inaccessible to light microscopy. Matrix components in most animals have also not yet been functionally tagged with fluorescent molecules to follow their dynamics in situ. Furthermore, genetic loss of many extracellular matrix components in animals leads to a cascade of diverse cell biological defects where the specific mechanism that initiated the perturbation is unclear (Rozario and DeSimone, 2010). Yet, advances in imaging techniques and the ability to perform complex genetic manipulations are helping to make progress in our understanding of the extracellular matrix in vivo. Here I present one example of how we are learning more about the cell biology of extracellular matrix from studies during development.I am particularly fascinated by the basement membrane, a thin, dense, cell-associated form of extracellular matrix that underlies epithelia and endothelia and surrounds fat, muscle, and Schwann cells (Yurchenco, 2011). The emergence of basement membrane coincided with the appearance of multicellularity in animals, suggesting that basement membranes were a prerequisite for formation of tissues and multicellular life (Ozbek et al., 2010; Hynes, 2012). Basement membranes are highly conserved and are composed of a core set of approximately six proteins or protein assemblies, including laminin, type IV collagen, perlecan, and nidogen. Work from cell culture and developing embryos have indicated that basement membranes are initially built on a polymeric network of secreted laminin molecules, which binds to sulfated glycolipids as well as integrin and dystroglycan receptors on the cell surface (Hohenester and Yurchenco, 2013). This laminin lattice serves as a template for the addition of other basement membrane components, including type IV collagen, which has the unique ability to self-associate with intermolecular covalent bonds that are thought to provide basement membranes with their tensile strength and stability (Khoshnoodi et al., 2008; Fidler et al., 2014).Basement membranes are generally thought of as stationary matrices that protect tissues from mechanical stresses, provide filtration and barrier functions, and act as a reservoir for growth factors (Yurchenco, 2011). Recent studies in visually accessible developmental systems, however, are revealing that basement membranes are dynamic scaffoldings that play instructive roles in tissue morphogenesis. For example, live imaging in Drosophila melanogaster using GFP-tagged type IV collagen has shown that tissue-specific regulation of basement membrane collagen has an important role in shaping numerous organs during development (Haigo and Bilder, 2011; Pastor-Pareja and Xu, 2011). Optical highlighting of laminin and type IV collagen in Caenorhabditis elegans larvae and collagen in cultured mouse salivary gland buds has also revealed that entire sheets of basement membrane move to facilitate tissue attachment and organ growth (Ihara et al., 2011; Harunaga et al., 2014; Matus et al., 2014). Work in developmental contexts has also shown regulated laminin deposition in coordinating polarized tissue formation and localized nidogen and perlecan accumulation in directing axon guidance and dendrite branching (Kim and Wadsworth, 2000; Rasmussen et al., 2012; Liang et al., 2015). Finally, work in my laboratory using tissue shifting and photobleaching of GFP-tagged laminin has identified a new adhesion system (B-LINK) that links neighboring tissues by connecting their adjacent basement membranes (Morrissey et al., 2014). These studies during development have uncovered the dynamic nature of basement membranes and the manner in which their remodeling actively directs specific cell behaviors and tissue formation events (summarized in Fig. 1).Open in a separate windowFigure 1.The basement membrane is a dynamic scaffold. During development, basement membranes assemble, grow, constrict tissues, and are actively remodeled to regulate diverse cellular behaviors and morphogenetic processes, including tissue polarity, tissue shaping, and tissue linkage.Developmental studies are poised to address many remaining fundamental questions on the cell biology of basement membranes. Basement membrane structure, composition, and assembly are still poorly understood and are primarily inferred from indirect biochemical and reconstitution studies. Developmental models will continue to be invaluable in validating biochemical studies. This is illustrated by recent work confirming the importance of the enzyme peroxidasin in creating sulfilimine cross-links in type IV collagen networks that are critical for basement membrane stability during fly, zebrafish, and C. elegans development (Gotenstein et al., 2010; Bhave et al., 2012; Fidler et al., 2014). In contrast, genetic loss of nidogen in C. elegans and mice, which was thought from biochemical analysis to be critical in bridging type IV collagen and laminin networks, has revealed that nidogen is not essential in connecting these two networks (Kim and Wadsworth, 2000; Hohenester and Yurchenco, 2013). In addition, live visualization of fluorescently tagged basement membrane components in transparent animals such as C. elegans and zebrafish will allow rapid and expanded forward genetic screens to determine mechanisms regulating basement membrane assembly and maintenance. As proteomic and expression profiling studies have revealed >200 matrix or matrix-associated basement membrane proteins, the complexity and regulation of basement membranes is likely vast (Manabe et al., 2008; Uechi et al., 2014).Developmental systems can also be used to address how basement membranes grow. This will be especially relevant with mechanically active tissues such as muscles. One experimentally accessible model is the C. elegans pharynx, a highly contractile organ (which beats ∼200 times/minute) encased in a basement membrane that expands dramatically during development (Fig. 2; Avery and Horvitz, 1989). How basement membranes balance tissue support, type IV collagen cross-linking, and dramatic expansion during development remains an open question.Open in a separate windowFigure 2.The basement membrane encasing the C. elegans foregut (pharynx) grows dramatically during development. (A) A differential interference contrast image of an embryo with the pharynx outlined. The pharynx is a basement membrane-encased contractile feeding organ that grinds and pumps food (bacteria) posteriorly into the intestine. (B and C) 3D-rendered isosurfaces of type IV collagen::mCherry show the approximately threefold increase in basement membrane surface area during pharyngeal growth from the L1 (B) to L4 (C) larval developmental stage. Bars, 5 µm. Images courtesy of R. Jayadev (Duke University, Durham, NC).An additional important area that requires further investigation is how basement membranes respond to physical forces. Analysis of mutants in genes encoding basement membrane components and a limited number of biophysical studies suggests that basement membranes mechanically support tissues (Pöschl et al., 2004; Candiello et al., 2007; Halfter et al., 2013). With the ability to visualize load across proteins with FRET-based tension sensors (Grashoff et al., 2010; Meng et al., 2011) it should be possible to probe individual basement membrane molecules in optically clear animals to identify components that support load and when these proteins experience mechanical stress (e.g., growth, tissue deformation, contractions).Live imaging of cell–basement membrane interactions during development will also allow a clearer understanding of the roles basement membrane components, associated growth factors, proteases, and receptors have in regulating diverse cellular behaviors (Hynes, 2009; Yurchenco, 2011). Thus, rather than complex endpoint phenotypes, live cell approaches allow examination of normal and disrupted cellular behaviors as they occur. For example, using live imaging we have shown that the integrin cell–matrix receptor has independent functions in both mediating cell–basement membrane attachment and establishing a specialized cell membrane domain that directs invasion through the basement membrane (Hagedorn et al., 2009; Wang et al., 2014).Finally, I expect that broad analysis of organismal development and physiology will continue to provide significant findings in cell–matrix biology. This is illustrated by studies showing dramatic changes in basement membrane accumulation during aging, and the findings that enhanced expression of matrix remodeling components increase organismal longevity (Candiello et al., 2010; Ewald et al., 2015). CRISPR/Cas9-mediated genome editing will also allow functional and live-cell analysis of cell–matrix interactions outside of current model systems, especially in basal metazoans such as translucent Cnidarian and Ctenophore embryos (Ikmi et al., 2014). These technical advances, and a wide experimental net, will bring a more comprehensive understanding of the fascinating, fundamental, and ancient interactions of cells and their surrounding extracellular matrix. This will have profound importance to human health, as numerous inherited diseases are caused by mutations in basement membrane components and changes in basement membrane structure are associated with the pathogenesis of diseases such as diabetes, hypertension, Alzheimer’s, and cancer (Tsilibary, 2003; Zlokovic, 2008; Van Agtmael and Bruckner-Tuderman, 2010; Lu et al., 2012; Kelley et al., 2014).     

Collagen family of proteins

Collagen molecules are structural macro-molecules of the extracellular matrix that include in their structure one or several domains that have a characteristic triple helical conformation. They have been classified by types that define distinct sets of polypeptide chains that can form homo- and heterotrimeric assemblies. All the collagen molecules participate in supramolecular aggregates that are stabilized in part by interactions between triple helical domains. Fourteen collagen types have been defined so far. They form a wide range of structures. Most notable are 1) fibrils that are found in most connective tissues and are made by alloys of fibrillar collagens (types I, II, III, V, and XI) and 2) sheets constituting basement membranes (type IV collagen), Descemet's membrane (type VIII collagen), worm cuticle, and organic exoskeleton of sponges. Other collagens, present in smaller quantities in tissues, play the role of connecting elements between these major structures and other tissue components. The fibril-associated collagens with interrupted triple helices (FACITs) (types IX, XII, and XIV) appear to connect fibrils to other matrix elements. Type VII collagen assemble into anchoring fibrils that bind epithelial basement membranes and entrap collagen fibrils from the underlying stroma to glue the two structures together. Type VI collagen forms thin-beaded filaments that may interact with fibrils and cells.     

Capillary endothelial cell cultures: phenotypic modulation by matrix components

Capillary endothelial cells of rat epididymal fat pad were isolated and cultured in media conditioned by bovine aortic endothelial cells and substrata consisting of interstitial or basement membrane collagens. When these cells were grown on interstitial collagens they underwent proliferation, formed a continuous cell layer and, if cultured for long periods of time, formed occasional tubelike structures. In contrast, when these cells were grown on basement membrane collagens, they did not proliferate but did aggregate and form tubelike structures at early culture times. In addition, cells grown on basement membrane substrata expressed more basement membrane constituents as compared with cells grown on interstitial matrices when assayed by immunoperoxidase methods and quantitated by enzyme-linked immunosorbent inhibition assays. Furthermore, when cells were grown on either side of washed, acellular amnionic membranes their phenotypes were markedly different. On the basement membrane surface they adhered, spread, and formed tubelike structures but did not migrate through the basement membrane. In contrast, when seeded on the stromal surface, these cells were observed to proliferate and migrate into the stromal aspect of the amnion and ultimately formed tubelike structures at high cell densities at longer culture periods (21 d). Thus, connective tissue components play important roles in regulating the phenotypic expression of capillary endothelial cells in vitro, and similar roles of the collagenous components of the extracellular matrix may exist in vivo following injury and during angiogenesis. Furthermore, the culture systems outlined here may be of use in the further study of differentiated, organized capillary endothelial cells in culture.     

Basement membrane protein distribution in LYVE-1-immunoreactive lymphatic vessels of normal tissues and ovarian carcinomas

The endothelial cells of blood vessels assemble basement membranes that play a role in vessel formation, maintenance and function, and in the migration of inflammatory cells. However, little is known about the distribution of basement membrane constituents in lymphatic vessels. We studied the distribution of basement membrane proteins in lymphatic vessels of normal human skin, digestive tract, ovary and, as an example of tumours with abundant lymphatics, ovarian carcinomas. Basement membrane proteins were localized by immunohistochemistry with monoclonal antibodies, whereas lymphatic capillaries were detected with antibodies to the lymphatic vessel endothelial hyaluronan receptor-1, LYVE-1. In skin and ovary, fibrillar immunoreactivity for the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1 was found in the basement membrane region of the lymphatic endothelium, whereas also heterogeneous reactivity for the laminin α5 chain was detected in the digestive tract. Among ovarian carcinomas, intratumoural lymphatic vessels were found especially in endometrioid carcinomas. In addition to the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1, carcinoma lymphatics showed immunoreactivity for the laminin α5 chain and Lutheran glycoprotein, a receptor for the laminin α5 chain. In normal lymphatic capillaries, the presence of primarily α4 chain laminins may therefore compromise the formation of endothelial basement membrane, as these truncated laminins lack one of the three arms required for efficient network assembly. The localization of basement membrane proteins adjacent to lymphatic endothelia suggests a role for these proteins in lymphatic vessels. The distribution of the laminin α5 chain and Lutheran glycoprotein proposes a difference between normal and carcinoma lymphatic capillaries.     

The role of collagen-derived proteolytic fragments in angiogenesis.

Basement membrane molecules and fragments derived from them are regulators of biological activities such as cell growth, differentiation and migration. This review describes proteolytically derived fragments from the non-collagenous (NC1) domain at the C-terminus of the basement membrane collagens type IV, XV and XVIII, which have been implicated as regulators of angiogenesis. Endostatin is an endogenous collagen XVIII/NC1 derivative, inhibiting endothelial cell proliferation and migration in vitro and tumor-growth in vivo. A homologous NC1 domain fragment of type XV collagen has anti-angiogenic activity as well. Furthermore, NC1 domain fragments of the most abundant basement membrane collagen, type IV collagen, have been shown to inhibit induced vessel growth.     

Epitope-defined monoclonal antibodies against multiplexin collagens demonstrate that type XV and XVIII collagens are expressed in specialized basement membranes

Type XV and type XVIII collagens are classified as part of multiplexin collagen superfamily and their C-terminal parts, endostatin and restin, respectively, have been shown to be anti-angiogenic in vivo and in vitro. The alpha1(XV) and alpha1(XVIII) collagen chains are reported to be localized mainly in the basement membrane zone, but their distributions in blood vessels and nonvascular tissues have yet to be thoroughly clarified. In the present study, we raised monoclonal antibodies against synthetic peptides of human alpha1(XV) and alpha1(XVIII) chains and used them for extensive investigation of the distribution of these chains. We came to the conclusion that nonvascular BMs contain mainly one of two types: subepithelial basement membranes that contained type XVIII in general, or skeletal and cardiac muscles that harbored mainly type XV. But basement membranes surrounding smooth muscle cells in vascular tissues contained one or both of them, depending on their locations. Interestingly, continuous capillaries contained both type XV and type XVIII collagens in their basement membranes; however, fenestrated or specialized capillaries such as glomeruli, liver sinusoids, lung alveoli, and splenic sinusoids expressed only type XVIII in their basement membranes, lacking type XV. This observation could imply that different functions of basement membranes in various tissues and organs use different mechanisms for the endogenous control of angiogenesis.     

Connective tissue biomatrix: its isolation and utilization for long- term cultures of normal rat hepatocytes

A new procedure is introduced for the isolation of connective tissue fibers, called biomatrix, containing a significant portion of the extracellular matrix (basement membrane components and components of the ground substance). Biomatrix isolated from normal rat liver contains >90% of the tissue's collagens and all of the known collagen types, including types I and III and basement membrane collagens. The purified collagenous fibers are associated with noncollagenous acidic proteins (including fibronectins and possibly small amounts of glycosaminoglycans). Procedures are also described for preparing tissue culture substrates with these fibers by either smearing tissue culture dishes with frozen sections or by shredding the biomatrix into small fibrils with a homogenizer. The biomatrix as a substrate has a remarkable ability to sustain normal rat hepatocytes long-term in culture. The hepatocytes, which on tissue culture plastic or on type I collagen gels do not survive more than a few weeks, have been maintained for more than 5 mo in vitro when cultured on biomatrix. These cells cultured on rat liver biomatrix show increased attachment and survival efficiencies, long-term survival (months) and retention of some hepatocyte-specific functions.     

The collagen family

Collagens are the most abundant proteins in mammals. The collagen family comprises 28 members that contain at least one triple-helical domain. Collagens are deposited in the extracellular matrix where most of them form supramolecular assemblies. Four collagens are type II membrane proteins that also exist in a soluble form released from the cell surface by shedding. Collagens play structural roles and contribute to mechanical properties, organization, and shape of tissues. They interact with cells via several receptor families and regulate their proliferation, migration, and differentiation. Some collagens have a restricted tissue distribution and hence specific biological functions.     

Properties of bovine nephritogenic antigen that induces anti-GBM nephritis in rats and its similarity to the Goodpasture antigen.

The nephritogenic antigen that induces antiglomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) in rats was isolated from collagenase-solubilized bovine renal basement membranes. Purification was achieved using antibody-coupled affinity columns which were originally used for the purification of trypsin-solubilized nephritogenic antigen (Sado et al. 1984a). The nephritogenic antigen was a heteropolymer composed of P2 (Mr 28 kDa) and P3 (Mr 30 kDa) polypeptides as monomers and their dimers in sodium-dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis. The P3 polypeptide was considered to be the nephritogenic epitope, since a fraction composed of the P2 polypeptide alone was not nephritogenic. The properties of the nephritogenic epitope were the same as those of the Goodpasture epitope (M2*), which is a noncollagenous domain of the alpha 3 chain of type IV collagen (Butkowski et al. 1985; Saus et al. 1988), indicating that the nephritogenic antigen is the same as the Goodpasture antigen.     

Current concepts of basement-membrane structure and function

Conclusion In this brief review we have attempted to describe the known components of basement membranes in relation to the morphology and function of these matrices. Further details of the molecular structures and biosynthesis of these components may be found in original papers and in various reviews (Kefalides, 1973; Spiro, 1976; Kefalides et al., 1979; Heathcote & Grant, 1981).Although basement membranes appear to contain essentially similar protein and carbohydrate moieties, the proportions and organization of these may differ and, in the opinion of the authors, the key to an understanding of basement membranes lies in the recognition of this heterogeneity. At present, structural models of basement membrane are far from satisfactory and should be regarded with reservation.