Four decades have passed since the first discovery of collagen IV by Kefalides in 1966. Since then collagen IV has been investigated extensively by a large number of research laboratories around the world. Advances in molecular genetics have resulted in identification of six evolutionary related mammalian genes encoding six different polypeptide chains of collagen IV. The genes are differentially expressed during the embryonic development, providing different tissues with specific collagen IV networks each having unique biochemical properties. Newly translated α-chains interact and assemble in the endoplasmic reticulum in a chain-specific fashion and form unique heterotrimers. Unlike most collagens, type IV collagen is an exclusive member of the basement membranes and through a complex inter-and intramolecular interactions form supramolecular networks that influence cell adhesion, migration, and differentiation. Collagen IV is directly involved in a number of genetic and acquired disease such as Alport's and Goodpasture's syndromes. Recent discoveries have also highlighted a new and direct role for collagen IV in the development of rare genetic diseases such as cerebral hemorrhage and porencephaly in infants and hemorrhagic stroke in adults. Years of intensive investigations have resulted in a vast body of information about the structure, function, and biology of collagen IV. In this review article, we will summarize essential findings on the structural and functional relationships of different collagen IV chains and their roles in health and disease.

The ␣-chains of the collagen superfamily are encoded with information that specifies self-assembly into fibrils, microfibrils, and networks that have diverse functions in the extracellular matrix. A key self-organizing step, common to all collagen types, is trimerization that selects, binds, and registers cognate ␣-chains for assembly of triple helical protomers that subsequently oligomerize into specific suprastructures. In this article, we review recent findings on the mechanism of chain selection and infer that terminal noncollagenous domains function as recognition modules in trimerization and are therefore key determinants of specificity in the assembly of suprastructures. This mechanism is also illustrated with computer-generated animations.Collagens are modular triple helical proteins that constitute the major structural components of the extracellular matrix of all animals. They occur as diverse suprastructures such as fibrils, microfibrils, and networks, which serve as self-organizing scaffolds for the attachment of other macromolecular complexes including laminin networks, proteoglycans, and cell surface receptors. The suprastructures play functional roles in cell adhesion, cell differentiation, tissue development, and the structural integrity of organs.Collagen suprastructures are assembled from a large family of gene products called ␣-chains. To date, 43 unique ␣-chains that belong to 28 types of collagens (types I-XXVIII) have been discovered in vertebrates. Based on their supramolecular architectures, they are further classified as fibril-forming, fibril-associated containing interrupted triple helices (FACIT), 3 beaded filament, anchoring fibril, network-forming, and transmembrane collagens (1-4). The assembly of all collagen suprastructures begins with the association of three type-specific ␣-chains (trimerization) that subsequently intertwine to form triple helical protomers, the building blocks of larger assemblies (1-5). Protomers are homotrimers or heterotrimers, composed of up to three different ␣-chains. They have in common at least one triple helical collagenous domain of varying length and two noncollagenous domains (NC) of variable sequence, size, and shape that are positioned at the N and C termini, designated herein as N-NC and C-NC domains, respectively. The terminal NC domains are excised, modified, or incorporated directly into the final suprastructure, depending on protomer type and function. Subsequently, specific protomers oligomerize into distinct suprastructures involving interactions that form end-to-end connections, lateral associations, and supercoiling of helices. Thus, protomer formation and oligomerization involve pivotal recognitions steps that target specific ␣-chains to assemble into a particular type of suprastructure.How the ␣-chains selectively recognize each other is a fundamental question in matrix biology that remains largely unanswered. Early studies on collagens I and III suggested that the C-NC domains play a critical role in the trimerization step that involv...

Nephrin is a type-1 transmembrane protein and a key component of the podocyte slit diaphragm, the ultimate glomerular plasma filter. Genetic and acquired diseases affecting expression or function of nephrin lead to severe proteinuria and distortion or absence of the slit diaphragm. Here, we showed by using a surface plasmon resonance biosensor that soluble recombinant variants of nephrin, containing the extracellular part of the protein, interact with each other in a specific and concentration-dependent manner. This molecular interaction was increased by twofold in the presence of physiological Ca 2؉ concentration, indicating that the binding is not dependent on, but rather promoted by Ca 2؉ . Furthermore, transfected HEK293 cells and an immortalized mouse podocyte cell line overexpressing full-length human nephrin formed cellular aggregates, with cell-cell contacts staining strongly for nephrin. The distance between plasma membranes at the nephrin-containing contact sites was shown by electron microscopy to be 40 to 50 nm, similar to the width of glomerular slit diaphragm. The cell contacts could be dissociated with antibodies reacting with the first two extracellular Ig-like domains of nephrin. Wild-type HEK293 cells were shown to express slit diaphragm components CD2AP, P-cadherin, FAT, and NEPH1. The results show that nephrin molecules exhibit homophilic interactions that could promote cellular contacts through direct nephrin-nephrin interactions, and that the other slit diaphragm components expressed could contribute to that interaction. Podocyte foot processes cover the outer aspect of the glomerular capillaries in an interdigitating manner. The slit between adjacent foot processes contains a highly ordered thin structure referred to as slit diaphragm (SD).The SD is thought to act as the ultimate albumin-excluding ultrafilter critical in the formation of primary urine.

Abstract. The expression pattern, subcellular localization, and the role of glycosylation of the human nephrin was examined in transfected cells. Stable cell lines, constitutively expressing a full-length human nephrin cDNA construct, were generated from transfected immortalized mouse podocytes (IMP) and a human embryonic kidney cell line (HEK-293). Immunofluorescence confocal microscopy of transfected cells showed plasma membrane localization of the recombinant nephrin. Immunoblotting showed that the recombinant nephrin expressed in transfected cell lines migrated as a double band with a molecular weight of 185 kD. When cells were treated with the N-glycosylation inhibitor, tunicamycin, the molecular weight of nephrin was decreased to a single immunoband of 150 kD, indicating that the shift in the electrophoretic migration of nephrin is due to N-linked carbohydrate moieties. It was further shown that this glycosylation process is highly sensitive to inhibition by tunicamycin, which is a naturally occurring antibiotic, leading to retention of nonglycosylated nephrin molecules in the endoplasmic reticulum. It was concluded that N-glycosylation of nephrin is crucial for its proper folding and thereby plasma membrane localization; therefore, inhibition of this process might be an important factor in the onset of pathogenesis of some acquired glomerular diseases.

The mechanism by which glucocorticoids govern antiproteinuric effect in nephrotic syndrome remains unknown. Present study examined the protective role of dexamethasone (DEX) in the intracellular trafficking of nephrin under endoplasmic reticulum (ER) stress. Human embryonic kidney-293 cell line expressing a full-length human nephrin was cultured in mediums containing 5.5 or 25 mM glucose with or without DEX. The result revealed that glucose starvation evoked a rapid ER stress leading to formation of underglycosylated nephrin that was remained in the ER as a complex with calreticulin/calnexin. DEX rescued this interfered trafficking through binding to its receptor and stimulating the mitochondrial transcripts and adenosine 5' triphosphate (ATP) production, leading to synthesis of fully glycosylated nephrin. These results suggest that ER-stress in podocytes may cause alteration of nephrin N-glycosylation, which may be an underlying factor in the pathomechanism of the proteinuria in nephrotic syndrome. DEX may restore this imbalance by stimulating expression of mitochondrial genes, resulted in the production of ATP that is essential factor for proper folding machinery aided by the ER chaperones.

Nephrin is a key functional component of the slit diaphragm, the structurally unresolved molecular filter in renal glomerular capillaries. Abnormal nephrin or its absence results in severe proteinuria and loss of the slit diaphragm. The diaphragm is a thin extracellular membrane spanning the approximately 40-nm-wide filtration slit between podocyte foot processes covering the capillary surface. Using electron tomography, we show that the slit diaphragm comprises a network of winding molecular strands with pores the same size as or smaller than albumin molecules, as demonstrated in humans, rats, and mice. In the network, which is occasionally stratified, immunogold-nephrin antibodies labeled individually detectable globular cross strands, about 35 nm in length, lining the lateral elongated pores. The cross strands, emanating from both sides of the slit, contacted at the slit center but had free distal endings. Shorter strands associated with the cross strands were observed at their base. Immunolabeling of recombinant nephrin molecules on transfected cells and in vitrified solution corroborated the findings in kidney. Nephrin-deficient proteinuric patients with Finnish-type congenital nephrosis and nephrin-knockout mice had only narrow filtration slits that lacked the slit diaphragm network and the 35-nm-long strands but contained shorter molecular structures. The results suggest the direct involvement of nephrin molecules in constituting the macromolecule-retaining slit diaphragm and its pores.

In patients with SRN, some missense mutations in the NPHS2 gene not only lead to misfolding and mislocalization of the mutated podocin, but they can also interfere with slit diaphragm structure and function by altering the proper trafficking of nephrin to the plasma membrane.

Collagens comprise a large superfamily of extracellular matrix proteins that play diverse roles in tissue function. The mechanism by which newly synthesized collagen chains recognize each other and assemble into specific triple-helical molecules is a fundamental question that remains unanswered. Emerging evidence suggests a role for the non-collagenous domain (NC1) located at the C-terminal end of each chain. In this study, we have investigated the molecular mechanism underlying chain selection in the assembly of collagen IV. Using surface plasmon resonance, we have determined the kinetics of interaction and assembly of the ␣1(IV) and ␣2(IV) NC1 domains. We show that the differential affinity of ␣2(IV) NC1 domain for dimer formation underlies the driving force in the mechanism of chain discrimination. Given its characteristic domain recognition and affinity for the ␣1(IV) NC1 domain, we conclude that the ␣2(IV) chain plays a regulatory role in directing chain composition in the assembly of (␣1) 2 ␣2 triple-helical molecule. Detailed crystal structure analysis of the [(␣1) 2 ␣2] 2 NC1 hexamer and sequence alignments of the NC1 domains of all six ␣-chains from mammalian species revealed the residues involved in the molecular recognition of NC1 domains. We further identified a hypervariable region of 15 residues and a ␤-hairpin structural motif of 13 residues as two prominent regions that mediate chain selection in the assembly of collagen IV. To our knowledge, this report is the first to combine kinetics and structural data to describe molecular basis for chain selection in the assembly of a collagen molecule.Collagens comprise a major superfamily of extracellular matrix (ECM) 3 proteins that play a key role in the structural integrity of all tissues. At least 27 different collagen types, consisting of 42 distinct gene products, have been identified in vertebrates, underlining their vast diversity in biological functions such as tissue compartmentalization and specialization during the development (1). In the endoplasmic reticulum, the newly synthesized collagen chains assemble into triple-helical molecules with specific chain compositions, which oligomerize to form supramolecular structures, including filaments and networks after secretion to the ECM.Some collagens are obligate homotrimers, such as collagen III, which comprises three identical pro-␣1(III) chains forming an ␣1(III) 3 procollagen, whereas others form heterotrimers containing at least one different ␣-chain; such as collagen I (2). Despite significant sequence identity and a propensity to form triple helices, procollagen chains have an extraordinary ability to discriminate between each other in the endoplasmic reticulum to form specific collagen types. For example, skin fibroblasts express six highly homologous but genetically distinct fibrilforming procollagen chains that are assembled in a type-specific manner to form type I, III, and V collagens. The mechanism by which different collagen chains are selected for assembly is a fundamental question...