Nicholas A. Kefalides

Biochemistry and Metabolism of Basement Membranes

Publisher Summary Basement membranes or basal laminae are ubiquitous in the vertebrate and invertebrate world and constitute important structural and functional extracellular matrices. Basement membranes are composed of fine fibrils, 40–60 A in diameter, arranged randomly in a granular matrix. Three general functions are ascribed to basement membranes: they (1) act as a semipermeable filter, (2) provide support, and (3) serve as a boundary between cell types or between cell layers and the underlying connective tissue. Basement membranes are insoluble in buffers at neutral pH, although small amounts of material can be brought into solution with acid buffers. Complete solubilization can be accomplished by reduction and alkylation in the presence of denaturant or after limited proteolysis. Collagen molecules are present in basement membranes. These apparently exist as triple-helical procollagen molecules composed of identical pro-α chains. Collagenous peptides equivalent in size to α chains can be isolated from digests of basement membranes. Noncollagenous glycoproteins are present in the matrix of most basement membranes; they are linked to the procollagen molecules via disulfide bonds and lysine- or hydroxylysine-derived cross-links.

Isolation of a collagen from basement membranes containing three identical α-chains

Abstract Chromatographic studies on CM-cellulose of collagens isolated from basement membranes of the glomerulus, lens capsule and Descemets membrane indicate that the molecule is composed of three identical α -1 chains. The M. Wt. of the α -1 chains from basement membranes is 108,000; it is higher than that of α -1 chains of interstitial collagens by an amount attributed to the excess hexose. Chemical analyses reveal high hydroxylysine, hydroxyproline and glycine content and a low amount of alanine. There are 8 residues of half-cystine. Total carbohydrate is about 12%, consisting of equimolar amounts of glucose and galactose.

Comparative adherence of granulocytes to endothelial monolayers and nylon fiber.

Adherence of granulocytes to tissue culture monolayers of endothelium averaged 26.2 +/- 1.3% SEM, which was similar to their adherence on 50-mg nylon fiber columns (27.7 +/- 3.6%). In contrast, adherence to epithelial cells, fibroblasts, kidney cells, and plastic Petri dishes without monolayers was only 12.4, 9.9, 11.1, and 4.3%, respectively. Cyclic nucleotides and adherence-modifying plasma factors induced changes of adherence to endothelium similar to those in nylon fiber columns. Adherence of granulocytes in whole blood was the same as for purified granulocytes in Hanks balanced salt solution. Exposure of endothelial monolayers to 0.18% trypsin for 10 min reduced subsequent granulocyte adherence to 25.2% of control values. Incubation of trypsin-treated monolayers with nutrient medium for 4 h did not improve adherence, but values returned to normal or above by 24 h, with or without serum proteins present in the nutrient medium. The similarity of granulocyte adherence to nylon fiber and to endothelial monolayers in vitro suggests that results with the nylon fiber assay reflect in vivo granulocyte-endothelium interaction. Furthermore, the endothelial monolayer offers a new model for studying this cell-cell relationship in vitro.

Biochemical properties of human glomerular basement membrane in normal and diabetic kidneys.

To determine the presence of any significant structural abnormalities in the glomerular basement membrane (GBM) of diabetic individuals, GBM from normal and diabetic human kidneys were isolated and analyzed chemically and structurally. The amino acid composition of the normal GBM revealed the presence of significant amounts of hydroxyproline, hydroxylysine, glycine, and carbohydrate suggesting the presence of a collagen-like protein. There was no significant increase in the amount of hydroxylysine, hydroxyproline, or in the hydroxylysine-linked glycoside glucosyl-galactose in the diabetic kidneys. There was, however, a significant decrease in the cystine and sialic acid content of GBM from diabetic kidneys. It was further shown that the alpha-chains isolated from the collagens of normal and diabetic basement membranes had similar amino acid and carbohydrate compositions. The hydroxylysine, hydroxyproline, glycine, and hexose contents were higher by 82, 56, 74, and 94%, respectively in the alpha-chains compared with the intact basement membranes from both the normal and diabetic kidneys. The results indicate that the slight increases in hydroxylysine and hexose content observed occasionally in diabetic GBM preparations are of no statistical significance and cannot be attributed to increases in the activities of enzymes which hydroxylate lysine or glycosylate hydroxylysine, respectively.

Virus Infection of Endothelial Cells Increases Granulocyte Adherence

Adherence of human granulocytes was measured on endothelial monolayers of human and bovine origin, grown in 35-mm Diam petri dishes and in cluster wells. Adherence to human endothelium in petri dishes using 1.0 ml of whole blood averaged 17.9+/-3.7%, and to bovine endothelium was 20.3+/-3.7%. Cluster wells required only 1/5 the endothelial cells needed for petri dishes, and 0.25 ml of whole blood yielded average adherence of 26.2+/-3.4 to human cells and 28.0+/-3.7 to bovine in the wells. The impact of infection of the endothelium by different viruses on subsequent granulocyte adherence was measured. Polio virus produced an acute lytic infection of human endothelial cells, with associated increased adherence to 185.4% of control 24 h after inoculation. Significantly increased adherence was noted at 6 h, before detectable cytopathic effect. Herpes simplex type I caused a similar rapidly lytic infection of bovine endothelium associated with increased adherence to 213.7% of control 6 h after inoculation. This augmented adherence could be demonstrated when granulocytes were suspended in physiologic saline solution, showing that antibody and complement need not be present. Trypsin treatment of infected monolayers did not prevent the augmentation, and supernate from infected monolayers increased the adherence of polymorphonuclear leukocytes to normal, uninfected monolayers. Chronic, slowly lytic infections, lasting 7 d or more, were induced with adenovirus in human endothelium and with measles virus in bovine cells. Adherence increased as virus was noted in the cell cultures on day 4, several days before cytotoxicity was seen. Thus, chronic viral infection of the endothelium appears possible, and results in increased granulocyte adherence. In naturally occurring disease, such an infection may act synergistically with adherent granulocytes to damage the endothelium, and may represent an in vitro model of vasculitis.

The embryonic rat parietal yolk sac: Changes in the morphology and composition of its basement membrane during development☆

Abstract The basement membrane (Reicherts membrane) of the entire capsular portion of the parietal yolk sac of rat embryos was examined both morphologically and chemically at various stages of gestation. The overall microscopic and compositional analyses showed Reicherts membrane to be typical of basement membranes isolated from other tissues and species. However, with increasing gestational age (from 11.5 to 17.5 days) a number of changes involving Reicherts membrane were noted: 1. The thickness increased rapidly then declined, while the surface area increased tenfold; 2. The total protein content increased twenty-fold while the collagen content increased eight-fold. As a result, the relative collagen content declined significantly; 3. The changes in the amino acid and carbohydrate composition were consistent with the latter finding. The observations listed above were evaluated in light of their possible relevance to an understanding of the morphogenesis of basement membranes during development, and to the possible mechanisms involved in pathogenesis of basement membrane dysfunction.

Isolation and characterization of cyanogen bromide peptides from basement membrane collagen

Abstract Twelve peptides were isolated and characterized after cyanogen bromide cleavage of the α-chain of basement membrane collagen from bovine lens capsule. Glycine accounted for about 1 3 of all amino acids in each peptide. 4-Hydroxyproline was present in all; 3-hydroxyproline, in 7, and hydroxylysine, in 9 peptides. 54 to 100% of the hydroxylysine in these peptides was substituted with glucosyl-galactose; only 1 contained galactose. One peptide contained 2 residues of mannose, 1 of glucosamine, 4 of half-cystine, 11 of hydroxylysine, and 7 of glucosyl-galactose. The molecular weight of the chain is 100033 without the hexose and 113600 including the hexose.

Isolation of laminin from human placental basement membranes: amnion, chorion and chorionic microvessels.

Laminin components were solubilized from basement membranes of amnion, chorion and chorionic microvessels of human placenta without prior protease digestion. These structures, after isolation, were initially processed in a sonicator bath containing a solution of Triton X-100, EDTA and 2M NaCl and the laminins extracted sequentially with 0.5M NaCl, 8 M urea, and 8 M urea + 2% 2-mercaptoethanol + 2% SDS. A high molecular weight (appr. 1 x 10(6)) complex containing laminins was purified by gel filtration on a Sepharose CL-2B column. This complex migrated as a single band on gel electrophoresis before reduction but resolved, after reduction, into four major laminin components, laminin A (350,000 M.W.), laminin M (240,000 M.W.) and laminins B1 and B2 (195,000 and 185,000 M.W.). Laminin M is a new molecular species of this protein.

Collagen crosslinks: Occurrence in basement membrane collagens

Abstract Basement membrane preparations from anterior lens capsule, Descemets membrane, and renal glomerulus were reduced with NaB3H4 in order to label carbonyl-derived crosslinks. Quantitative incorporation of tritium into the basement membranes was found, similar to the levels observed in fibrous collagens. A considerable proportion of the label was chromatographically identical with the reduced aldehydes and crosslinks of collagen, suggesting that is is the collagenous portions of basement membrane which contain these compounds. This was substantiated by showing that the isolated collagenous proteins contained substantial amounts of reduced aldehydes and crosslinks.

Isolation of the target antigen of human anti-tubular basement membrane antibody-associated interstitial nephritis.

Using a monoclonal anti-tubular basement membrane antibody (alpha TBM-Ab) affinity column, we isolated from collagenase-solubilized human renal tissue (HSRTA) a predominantly 48,000-mol-wt moiety (H3M-1) which is selectively recognized by antisera from two patients with alpha TBM-Ab-associated interstitial nephritis (alpha TBM disease). Whereas both antisera had alpha TBM-Ab titers of 1:64-1:128 by immunofluorescence on tissue sections, their reactivity with H3M-1 in a solid-phase radioimmunoassay was demonstrable at dilutions up to 1:10,000. While these sera displayed some reactivity with pre-column HSRTA, this was markedly less than with H3M-1. HSRTA depleted of H3M-1 by passage over the alpha TBM-Ab affinity column was almost completely depleted of reactivity. Neither pooled normal human sera nor sera from patients with a variety of renal lesions not associated with alpha TBM-Ab (including interstitial nephritis and antiglomerular basement membrane disease) were reactive with H3M-1. Both patient antisera containing alpha TBM-Ab were also highly reactive with R3M-1, the 48,000-mol-wt rabbit glycoprotein antigen of experimental alpha TBM disease. Furthermore, a competitive inhibition radioimmunoassay revealed that alpha TBM-Ab from rodents with experimental alpha TBM disease could inhibit 45-98% of the R3M-1 binding reactivity of patient antisera and 85% of the H3M-1 binding reactivity of patient antisera, thus suggesting paratypic cross-reactivity. We conclude, therefore, that tubular basement membrane target epitopes and their paratypic recognition are highly conserved among mammals.