Yoshikazu Sado

Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different α chains of human type IV collagen

A group of rat monoclonal antibodies recognizing the six different α chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen α chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of α chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.

Distinct target-derived signals organize formation, maturation, and maintenance of motor nerve terminals

Target-derived factors organize synaptogenesis by promoting differentiation of nerve terminals at synaptic sites. Several candidate organizing molecules have been identified based on their bioactivities in vitro, but little is known about their roles in vivo. Here, we show that three sets of organizers act sequentially to pattern motor nerve terminals: FGFs, beta2 laminins, and collagen alpha(IV) chains. FGFs of the 7/10/22 subfamily and broadly distributed collagen IV chains (alpha1/2) promote clustering of synaptic vesicles as nerve terminals form. beta2 laminins concentrated at synaptic sites are dispensable for embryonic development of nerve terminals but are required for their postnatal maturation. Synapse-specific collagen IV chains (alpha3-6) accumulate only after synapses are mature and are required for synaptic maintenance. Thus, multiple target-derived signals permit discrete control of the formation, maturation, and maintenance of presynaptic specializations.

Type IV Collagen of the Glomerular Basement Membrane EVIDENCE THAT THE CHAIN SPECIFICITY OF NETWORK ASSEMBLY IS ENCODED BY THE NONCOLLAGENOUS NC1 DOMAINS

The ultrafiltration function of the glomerular basement membrane (GBM) of the kidney is impaired in genetic and acquired diseases that affect type IV collagen. The GBM is composed of five (α1 to α5) of the six chains of type IV collagen, organized into an α1·α2(IV) and an α3·α4·α5(IV) network. In Alport syndrome, mutations in any of the genes encoding the α3(IV), α4(IV), and α5(IV) chains cause the absence of the α3·α4·α5 network, which leads to progressive renal failure. In the present study, the molecular mechanism underlying the network defect was explored by further characterization of the chain organization and elucidation of the discriminatory interactions that govern network assembly. The existence of the two networks was further established by analysis of the hexameric complex of the noncollagenous (NC1) domains, and the α5 chain was shown to be linked to the α3 and α4 chains by interaction through their respective NC1 domains. The potential recognition function of the NC1 domains in network assembly was investigated by comparing the composition of native NC1 hexamers with hexamers that were dissociated and reconstituted in vitro and with hexamers assembled in vitro from purified α1-α5(IV) NC1 monomers. The results showed that NC1 monomers associate to form native-like hexamers characterized by two distinct populations, an α1·α2 and α3·α4·α5 heterohexamer. These findings indicate that the NC1 monomers contain recognition sequences for selection of chains and protomers that are sufficient to encode the assembly of the α1·α2 and α3·α4·α5 networks of GBM. Moreover, hexamer formation from the α3, α4, and α5 NC1 monomers required co-assembly of all three monomers, suggesting that mutations in the NC1 domain in Alport syndrome may disrupt the assembly of the α3·α4·α5 network by interfering with the assembly of the α3·α4·α5 NC1 hexamer.

Glomerular Basement Membrane IDENTIFICATION OF A NOVEL DISULFIDE-CROSS-LINKED NETWORK OF α3, α4, AND α5 CHAINS OF TYPE IV COLLAGEN AND ITS IMPLICATIONS FOR THE PATHOGENESIS OF ALPORT SYNDROME

Glomerular basement membrane (GBM) plays a crucial function in the ultrafiltration of blood plasma by the kidney. This function is impaired in Alport syndrome, a hereditary disorder that is caused by mutations in the gene encoding type IV collagen, but it is not known how the mutations lead to a defective GBM. In the present study, the supramolecular organization of type IV collagen of GBM was investigated. This was accomplished by using pseudolysin (EC3.4.24.26) digestion to excise truncated triple-helical protomers for structural studies. Two distinct sets of truncated protomers were solubilized, one at 4 °C and the other at 25 °C, and their chain composition was determined by use of monoclonal antibodies. The 4 °C protomers comprise the α1(IV) and α2(IV) chains, whereas the 25 °C protomers comprised mainly α3(IV), α4(IV), and α5(IV) chains along with some α1(IV) and α2(IV) chains. The structure of the 25 °C protomers was examined by electron microscopy and was found to be characterized by a network containing loops and supercoiled triple helices, which are stabilized by disulfide cross-links between α3(IV), α4(IV), and α5(IV) chains. These results establish a conceptual framework to explain several features of the GBM abnormalities of Alport syndrome. In particular, the α3(IV)·α4(IV)·α5(IV) network, involving a covalent linkage between these chains, suggests a molecular basis for the conundrum in which mutations in the gene encoding the α5(IV) chain cause defective assembly of not only α5(IV) chain but also the α3(IV) and α4(IV) chains in the GBM of patients with Alport syndrome.

Basement membrane abnormalities in human eyes with diabetic retinopathy.

Vascular and parenchymal basement membranes (BMs) are thickened in diabetes, but alterations in individual BM components in diabetic eyes, especially in diabetic retinopathy (DR), are obscure. To identify abnormalities in the distribution of specific constituents, we analyzed cryostat sections of human eyes obtained at autopsy (seven normal, five diabetic without DR, and 13 diabetic with DR) by immunofluorescence with antibodies to 30 BM and extracellular matrix components. In non-DR eyes, no qualitative changes of ocular BM components were seen. In some DR corneas, epithelial BM was stained discontinuously for laminin-1, entactin/nidogen, and alpha3-alpha4 Type IV collagen, in contrast to non-DR corneas. Major BM alterations were found in DR retinas compared to normals and non-DR diabetics. The inner limiting membrane (retinal BM) of DR eyes had accumulations of fibronectin (including cellular) and Types I, III, IV (alpha1-alpha2), and V collagen. The BM zone of new retinal blood vessels in neovascularized areas accumulated tenascin and Type XII collagen, whereas normal, diabetic, and adjacent DR retinas showed only weak and irregular staining. In preretinal membranes, perlecan, bamacan, and Types VI, VIII, XII, and XIV collagen were newly identified. Diabetic BM thickening appears to involve qualitative alterations of specific BM markers at an advanced disease stage, with the appearance of DR.

Human Corneal Epithelial Basement Membrane and Integrin Alterations in Diabetes and Diabetic Retinopathy1

Corneas of diabetic patients have abnormal healing and epithelial adhesion, which may be due to alterations of the corneal extracellular matrix (ECM) and basement membrane (BM). To identify such alterations, various ECM and BM components and integrin receptors were studied by immunofluorescence on sections of normal and diabetic human corneas. Age-matched corneas from 15 normal subjects, 10 diabetics without diabetic retinopathy (DR), and 12 diabetics with DR were used. In DR corneas, the composition of the central epithelial BM was markedly altered, compared to normal or non-DR diabetic corneas. In most cases the staining for entactin/nidogen and for chains of laminin-1 (α1β1γ1) and laminin-10 (α5β1γ1) was very weak, discontinuous, or absent over large areas. Other BM components displayed less frequent changes. The staining for α3β1 (VLA-3) laminin binding integrin was also weak and discontinuous in DR corneal epithelium. Components of stromal ECM remained unchanged even in DR corneas. Therefore, distinct changes were identified in the composition of the epithelial BM in DR corneas. They may be due to increased degradation or decreased synthesis of BM components and related integrins. These alterations may directly contribute to the epithelial adhesion and wound healing abnormalities found in diabetic corneas.

Mouse Model of X-Linked Alport Syndrome

X-linked Alport syndrome (XLAS) is a progressive disorder of basement membranes caused by mutations in the COL4A5 gene, encoding the alpha5 chain of type IV collagen. A mouse model of this disorder was generated by targeting a human nonsense mutation, G5X, to the mouse Col4a5 gene. As predicted for a nonsense mutation, hemizygous mutant male mice are null and heterozygous carrier female mice are mosaic for alpha5(IV) chain expression. Mutant male mice and carrier female mice are viable through reproductive age and fertile. Mutant male mice died spontaneously at 6 to 34 wk of age, and carrier female mice died at 8 to 45 wk of age, manifesting proteinuria, azotemia, and progressive and manifold histologic abnormalities of the kidney glomerulus and tubulointerstitium. Ultrastructural abnormalities of the glomerular basement membrane, including lamellation and splitting, were characteristic of human XLAS. The mouse model described here recapitulates essential clinical and pathologic findings of human XLAS. With alpha5(IV) expression reflecting X-inactivation patterns, it will be especially useful in studying determinants of disease variability in the carrier state.

Transcriptome-based systematic identification of extracellular matrix proteins

Extracellular matrix (ECM), which provides critical scaffolds for all adhesive cells, regulates proliferation, differentiation, and apoptosis. Different cell types employ customized ECMs, which are thought to play important roles in the generation of so-called niches that contribute to cell-specific functions. The molecular entities of these customized ECMs, however, have not been elucidated. Here, we describe a strategy for transcriptome-wide identification of ECM proteins based on computational screening of >60,000 full-length mouse cDNAs for secreted proteins, followed by in vitro functional assays. These assays screened the candidate proteins for ECM-assembling activities, interactions with other ECM molecules, modifications with glycosaminoglycans, and cell-adhesive activities, and were then complemented with immunohistochemical analysis. We identified 16 ECM proteins, of which seven were localized in basement membrane (BM) zones. The identification of these previously unknown BM proteins allowed us to construct a body map of BM proteins, which represents the comprehensive immunohistochemistry-based expression profiles of the tissue-specific customization of BMs.

Cellular and subcellular immunolocalization of ClC-5 channel in mouse kidney: colocalization with H+-ATPase.

To determine the immunolocalization of ClC-5 in the mouse kidney, we developed a ClC-5-specific rat monoclonal antibody. Immunoblotting demonstrated an 85-kDa band of ClC-5 in the kidney and ClC-5 transfected cells. Immunocytochemistry revealed significant labeling of ClC-5 in brush-border membrane and subapical intracellular vesicles of the proximal tubule. In addition, apical and cytoplasmic staining was observed in the type A intercalated cells in the cortical collecting duct. In contrast, the staining was minimal in the outer and inner medullary collecting ducts and the thick ascending limb. Western blotting of vesicles immunoisolated by the ClC-5 antibody showed the presence of H+-ATPase, strongly indicating that these two proteins were present in the same membranes. Double labeling with antibodies against ClC-5 and H+-ATPase and analysis by confocal images showed that ClC-5 and H+-ATPase colocalized in these ClC-5-positive cells. These findings suggest that ClC-5 might be involved in the endocytosis and/or the H+ secretion in the proximal tubule cells and the cortical collecting duct type A intercalated cells in mouse kidney.

A New Model of Development of the Mammalian Ovary and Follicles

Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.