Milton E. Noelken

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.

Seminiferous Tubule Basement Membrane COMPOSITION AND ORGANIZATION OF TYPE IV COLLAGEN CHAINS, AND THE LINKAGE OF α3(IV) AND α5(IV) CHAINS

Seminiferous tubule basement membrane (STBM) plays an important role in spermatogenesis. In the present study, the composition and structural organization of type IV collagen of bovine STBM was investigated. STBM was found to be composed of all six α-chains of type IV collagen based upon immunocytochemical and biochemical analysis. The content of α3(IV) chain (40%) and the α4(IV) chain (18%) was substantially higher than in any other basement membrane collagen. The supramolecular structure of the six α(IV) chains was investigated using pseudolysin (EC 3.4.24.26) digestion to excise triple-helical molecules, subsequent collagenase digestion to produce NC1 hexamers and antibody affinity chromatography to resolve populations of NC1 hexamers. The hexamers, which reflect specific arrangements of α(IV) chains, were characterized for their α(IV) chain composition using high performance liquid chromatography, two-dimensional electrophoresis, and immunoblotting with α(IV) chain-specific antibodies. Three major hexamer populations were found that represent the classical network of the α1(IV) and α2(IV) chains and two novel networks, one composed of the α1(IV)-α6(IV) chains and the other composed of the α3(IV)-α6(IV) chains. The results establish a structural linkage between the α3(IV) and α5(IV) chains, suggesting a molecular basis for the conundrum in which mutations in the gene encoding the α5(IV) chain cause defective assembly of the α3(IV) chain in the glomerular basement membrane of patients with Alport syndrome.

Goodpasture Syndrome: Molecular Architecture and Function of Basement Membrane Antigen

In studies of victims from the influenza pandemic of 1918–1919, Ernest Goodpasture described the coexistence of fatal pulmonary hemorrhage and proliferative glomerulonephritis in a young man (25). The term Good- pasture syndrome was coined by Stanton and Tange in 1958 to describe cases characterized by the coexistence of these manifestations (73). The syndrome is now defined as an autoimmune disorder consisting of the triad of glomerulonephritis, lung hemorrhage, and antiglomerular basement membrane antibody formation, and it includes a broad spectrum of clinical features, ranging from massive pulmonary hemorrhage with little overt evidence of renal disease to fulminant crescentic glomerulonephritis and little overt evidence of pulmonary hemorrhage (24). Although the etiology of this syndrome remains unknown, profound advances have been made during the last 25 years in delineating the pathogenesis of the glomerulonephritis.

Physical properties of collagen--sodium dodecyl sulfate complexes.

Sodium dodecyl sulfate (NaDodSO4)--polyacrylamide gel electrophoresis and gel filtration chromatography of protein--NaDodSO4 complexes are frequently used to characterize collagen-like polypeptide components in mixtures obtained from extracts of basement membranes. However, electrophoresis yields anomalously high apparent molecular weights for collagenous polypeptides when typical globular proteins are used as molecular weight standards, and the use of gel filtration chromatography for this purpose was suspect because Nozaki et al. [Nozaki, Y., Schechter, N. M., Reynolds, J. A., & Tanford, C. (1976) Biochemistry 15, 3884--3890] found that asymmetric particles, including NaDodSO4--protein complexes, coeluted with native globular proteins of lower Stokes radius, when Sepharose 4B was used. To understand these effects and to improve the characterization of collagenous polypeptides, we investigated the secondary structure of NaDodSO4--collagen complexes with the use of circular dichroism, measured the NaDodSO4 content, studied the dependence of electrophoretic mobility on gel concentration, and extended work on gel filtration by use of a more porous gel, Sepharose CL-4B. We found that the anomalous behavior of collagen chains on NaDodSO4--polyacrylamide gel electrophoresis is due in large part to treatment of data and that the method can be used to determine rather accurate values for the number of residues per polypeptide chain. Our gel filtration results indicated that reliable molecular weights can be obtained when Sepharose CL-4B is used. These methods can be applied equally well to collagenous and noncollagenous polypeptides.

[19] Estimation of the size of collagenous proteins by electrophoresis and gel chromatography☆

Abstract Collagenous polypeptides have apparent low electrophoretic mobilities compared to those of typical globular proteins when their molecular weights are examined as a function of mobility. If, however, the number of amino acid residues epr polypeptide chain is examined as a function of electrophoretic mobility, both collagenous polypeptides and globular proteins obey the same empirical linear relationship. The relationship suggests that the number of residues in collagenous polypeptides from sources such as basement membranes, which may contain both collagenous and noncollagenous regions, may be estimated accurately from SDS-gel electrophoresis. Calf skin collagen and its naturally occurring cross-linked multimers, as well as polypeptides derived from it, provide useful molecular standards in a range from 150 residues to 11,500 residues per polypeptide chain (corresponding to a molecular weight range from 13,500 to about 1,000,000 for collagen). The number of residues can in turn be translated into molecular weight values with a knowledge of the mean residue weight of the polypeptide of interest. In contrast, the molecular weight of collagenous chains can be directly estimated by gel chromatography in Sepharose CL-4B, since collagen chains give the correct molecular weight when compared to globular protein standards.

Estimation of the size of collagenous polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis☆

Abstract Interpretation of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis results for polypeptides which contain both collagenous and noncollagenous regions may be somewhat ambiguous since collagenous chains obey a different molecular weight vs mobility relationship than reduced globular proteins. In a recent study [Freytag, J. W., Noelken, M. E., and Hudson, B. G., 1979, Biochemistry18, 4761–4768], however, it was found that the α chains of calf skin collagen obeyed the same size-mobility relationship as reduced globular proteins when the number of residues was used as a measure of size. We extended that study over a broad size range and found the same result for 581 to 2104 residue polypeptides when 5% gels were used, and for 217 to 1052 residue polypeptides with 9% gels. On the other hand, SDS complexes of collagenous chains having fewer than 300 residues migrated considerably more slowly through 12.5% gels than their counter-parts from globular proteins. Also, SDS complexes of αs1-, β-, and γ2-casein which have 8.5, 16.7, and 20 mol% proline, respectively, had mobilities between those of SDS complexes of collagenous polypeptides and their reduced globular protein counterparts with the same number of residues. Our results indicate that SDS-polyacrylamide electrophoresis can be used to determine accurately the number of residues of collagenous polypeptides in the 217 to 2104 residue size range if appropriate gel concentrations are used. However, this conclusion does not apply to high-proline polypeptides in general.

Type IV collagen of engelbreth-holm-swarm tumor matrix : identification of constituent chains

The noncollagenous hexamer (NC1) domain of type IV collagen from Engelbreth-Holm-Swarm (EHS) sarcoma matrix was subjected to electrophoretic, amino-terminal amino acid sequence, and immunochemical analysis to determine which of the five known kinds of alpha(IV) chains are present. Electrophoretic analysis, whether by one-dimensional or two-dimensional electrophoresis, showed that nonlathyritic and lathyritic hexamer gave nearly identical patterns. Amino-terminal amino acid sequence analysis of hexamer subunits, transblotted from two-dimensional gels, revealed that the hexamer subunits were derived exclusively from the alpha 1 and alpha 2 chains. Western blots of hexamer subunits confirmed the sequence results, as the subunits. identified as alpha 1(IV) and alpha 2(IV) NC1 domains reacted with antibodies directed specifically against those subunits. Conversely, no reactivity of NC1 hexamer subunits was seen with Goodpasture serum, or with antibodies directed specifically against the alpha 3, alpha 4, and alpha 5 NC1 domains, confirming the lack of alpha 3, alpha 4, and alpha 5 chains. These results revealed that the type IV collagen component of the EHS sarcoma matrix is comprised exclusively of alpha 1 and alpha 2 chains. Its relative homogeneity simplifies, but restricts, interpretation of studies that employ it as a model type IV collagen because the studies would be based only on alpha 1 and alpha 2 chains.

Structure and composition of type IV collagen of bovine aorta

To determine the chain composition of type IV collagen of bovine thoracic aorta, we analyzed collagenase-solubilized carboxyl-terminal noncollagenous (NC1)-domains by high-pressure liquid chromatography, two-dimensional electrophoresis, immunoblotting and enzyme-linked immunoassay. In addition to the classical alpha 1- and alpha 2-chains, we found small amounts of the recently discovered alpha 3-, alpha 4- and alpha 5-chains. The alpha 3- and alpha 4-chains were, collectively, 7-13% of the total, and the alpha 5-chain was present in a low amount. Seventy-nine percent of the NC1-domains were dimerized. Immunolocalization studies on sections of aorta showed that the alpha 3- and alpha 5-chains were present, along with alpha 1- and alpha 2-chains, in the subendothelium and media. In capillaries of the media, the alpha 3-chain was found at relatively high levels and was co-localized with alpha 1- and alpha 2-chains. Digestion of aorta with Pseudomonas aeruginosa elastase yielded soluble multimolecular assemblies of type IV collagen. Electron microscopy results provided a direct demonstration of the supramolecular structure, in which the collagen molecules were tetramerized at the amino-terminal end and dimerized at the carboxyl-terminal end.

Studies on the subunit structure of thermostable glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus☆

Abstract Sedimentation equilibrium studies of the thermophilic glyceraldehyde-3-phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate: NAD oxidoreductase (phosphorylating), EC 1.2.1.12) in 5.0 m guanidine-HCl showed that the enzyme is dissociated into monodisperse subunits having a molecular weight of 36, 000 ± 1,200. Based on the molecular weight of the undissociated enzyme, it is concluded that it exists as a tetramer in the native state. C-terminal amino acid analyses revealed a single-type amino acid, leucine, suggesting identical subunits. In kinetic studies, the thermophilic enzyme showed marked resistance to enzymic inactivation by 8.0 m urea at mesophilic temperatures, but is rapidly inactivated in this solvent at thermophilic temperatures; some reversibility is detectable at all temperatures. However, in guanidine-HCl, the thermophilic enzyme is rapidly and irreversibly inactivated at all temperatures studied. Optical rotatory dispersion studies showed that the thermophilic enzyme is extensively unfolded in these denaturing solvents at 30 ° and above. In contrast, heating the enzyme in water resulted in an alteration in secondary structure that was concomitant with an increase in enzymic activity. The possible mechanism of inactivation by urea and guanidine-HCl is discussed.