Fibril-forming collagens in lamprey

Five types of collagen with triple-helical regions approximately 300 nm in length were found in lamprey tissues which show characteristic D-periodic collagen fibrils. These collagens are members of the fibril forming family of this primitive vertebrate. Lamprey collagens were characterized with respect to solubility, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carboxylmethyl-cellulose chromatography, peptide digestion patterns, composition, susceptibility to vertebrate collagenase, thermal stability, and segment long spacing-banding pattern. Comparison with fibril-forming collagens in higher vertebrates (types I, II, III, V, and XI) identified three lamprey collagens as types II, V, and XI. Both lamprey dermis and major body wall collagens had properties similar to type I but not the typical heterotrimer composition. Dermis molecules had only alpha 1(I)-like chains, while body wall molecules had alpha 2(I)-like chains combined with chains resembling lamprey type II. Neither collagen exhibited the interchain disulfide linkages or solubility properties of type III. The conservation of fibril organization in type II/type XI tissues in contrast to the major developments in type I and type III tissues after the divergence of lamprey and higher vertebrates is consistent with these results. The presence of type II and type I-like molecules as major collagens and types V and XI as minor collagens in the lamprey, and the differential susceptibility of these molecules to vertebrate collagenase is analogous to the findings in higher vertebrates.     

Folding defects in fibrillar collagens

Fibrillar collagens have a long triple helix in which glycine is in every third position for more than 1000 amino acids. The three chains of these molecules are assembled with specificity into several different molecules that have tissue-specific distribution. Mutations that alter folding of either the carboxy-terminal globular peptides that direct chain association, or of the regions of the triple helix that are important for nucleation, or of the bulk of the triple helix, all result in identifiable genetic disorders in which the phenotype reflects the region of expression of the genes and their tissue-specific distribution. Mutations that result in changed amino-acid sequences in any of these regions have different effects on folding and may have different phenotypic outcomes. Substitution for glycine residues in the triple helical domains are among the most common effects of mutations, and the nature of the substituting residue and its location in the chain contribute to the effect on folding and also on the phenotype. More complex mutations, such as deletions or insertions of triple helix, also affect folding, probably because of alterations in helical pitch along the triple helix. These mutations all interfere with the ability of these molecules to form the characteristic fibrillar array in the extracellular matrix and many result in intracellular retention of abnormal molecules.     

Current viewpoint on structure and on evolution of collagens. II. Fibril-associated collagens

Fibril-associated collagens (FACITs) form one of subfamilies included in family of collagens. Being minor components of connective tissue of multicellular animals, FACITs play an important role in structurization of extracellular matrix whose peculiarities determine essential intertissue differences. FACITs participate in regulation of sizes of banded collagen fibrils as well as are connecting links between various components extracellular matrix and cells in different tissues. Functional characteristics of FACIT molecules are determined by peculiarities of structural organization of their α-chains (breakdowns in collagenous domains and module structure of N-terminal noncollagenous sites), trimeric molecules (domains of trimerization) and supramolecular assemblies (mainly association with banded collagen fibrils and the inability to form homopolymeric supramolecular aggregates). The problem of evolution of this group of collagen molecules is also discussed. A hypothetical model of structural changes leading to formation of the FACIT subfamily is proposed.     

A current viewpoint on structure and evolution of collagens. I. Fibrillar collagens

This review summarizes current data of structure of the most representative group of superfamily of collagens—fibrillar collagens. The attention is focused on structural organization of individual domains and their functional role in the hierarchical stacking of collagen α-chains. There are presented characteristics of the main stages of biosynthesis and the supramolecular processing of fibrillar collagens. Also considered are some aspects of evolution of fibrillar collagens. The role of duplication of genome and genes, intergene combination, and translocation of exons in evolution of collagen genes is discussed.     

A modern viewpoint on structure and evolution of collagens. I. Fibrillar collagens

This review summarizes current data on structure of the most representative group of the collagen family--fibrillar collagens. Attention has been focused on structural organization of individual domains and their functional role in the hierarchical stacking of alpha-chains of collagens. There is presented characteristics of the main stages of biosynthesis and of supramolecular processing of fibrillar collagens. Also considered are some aspects of evolution of fibrillar collagens. The role of duplication of genome and genes, intergene rearrangements, and exon shuffling in evolution of collagen genes is discussed.     

Intermolecular interactions in type I collagens.

X-PLOR modelling of collagen dimers containing Gly-Glu-Arg in each chain has been carried out. The interaction between molecules when two Gly-Glu-Arg are present on each chain is found to be substantially less than two times that obtained with one per chain, implying that relative tilting of two collagen molecules does not offset the disadvantages of misalignment of the interacting moieties. This implies that if multiple (Glu(-)-Arg+)3 interactions are important in fibril formation, their lateral separations must be large enough to insure that they act independently.     

Light microscopic distinction of collagens in hepatic cirrhosis

Summary Pollen DNAse of Chlorophytum comosum was demonstrated cytochemically by coupling the breakdown of DNA (due to the endogenous DNAse) with acid phosphatase supplied to the reaction mixture. Precipitation of the free phosphate groups (from acid phosphatase reaction on the freed nucleotides) as lead phosphate results in a electron dense deposit, visible with the electron microscope. Enzyme activity using this technique was localized in vesicles along the intine (principally along the aperture region) and around the generative cell.     

Enzymes converting procollagens to collagens

Conversion from procollagen to collagen is a specific process that is a requirement for proper alignment of collagen molecules to form functional fibers. This process is catalyzed by at least three structurally and functionally distinct enzymes cleaving collagen types I-III. The cleavage processes possibly taking place in the more recently discovered collagen types are not known to any extent at this time. Two amino-terminal proteinases, one cleaving type I and type II procollagens and the other cleaving type III procollagen, have been purified close to homogeneity, and the more unspecific activity of carboxy-terminal proteinase has been isolated from several tissues. In our experimental model, however, cleavage of the carboxy-terminal propeptides of types I and III procollagen is differently affected by lysine. This suggests the presence of at least two distinct enzymes for the removal of carboxyl-terminal propeptides. The regulation of the reaction process from procollagen to collagen is not well known at present. The importance of the phenomenon in terms of fibril formation, however, is demonstrated by several elegant studies in vitro; and certain genetic disorders in which this process is defective demonstrate the significance in vivo. Moreover, the factors shown to effect the cleavage process may be potentially beneficial in the treatment of the pathological processes with abnormal collagen accumulation such as fibrosis. In this paper we briefly review the current knowledge of the converting enzymes, including some very recent findings of our laboratory as well as the evidence presented for the biological significance of the conversion process.     

Precipitation of collagens by polyethylene glycols

Types I, II, and III collagens are readily precipitated at neutral pH by polyethylene glycols (PEG). As the molecular weight fraction of the polyethylene glycols increases, they become more effective as precipitants on a weight basis. The amount of PEG required for precipitation depends on the pH, the ionic strength, and the nature of the buffer or salts present. In tissue culture media, low concentrations of collagens and procollagens may be quantitatively precipitated and readily collected by low-speed centrifugation. Polyethylene glycol precipitation can be used to obtain collagens and procollagens from tissue culture media at either analytical or preparative scale, and since the polyethylene glycols do not bind to collagens, the precipitates may be further analyzed directly by chromatographic or electrophoretic methods.     

Electrophoresis and electroblotting of native collagens

Electrophoretic and immunoblotting techniques, while now used routinely for the biochemical characterization of many proteins, have not been used for the identification of native collagens. We present here an acidic electrophoresis system using very low percentage acrylamide gels which maintains collagen solubility and allows migration of native dermal collagens. The method gives uniform gels which can be made mechanically stable for subsequent electroblotting. The resulting nitrocellulose transfer allows immunological detection of collagens using either polyclonal or monoclonal antibodies and can be used to screen antibody specificities. The majority of murine monoclonal antibodies directed against collagen bind only to conformational epitopes on the native triple-helical collagen, and thus cannot be screened by Western blotting. This method therefore enables the electrophoretic screening of these monoclonal antibodies and provides an alternative approach for their characterization.     

Microplate reader-based quantitation of collagens.

By using a picrosirius dye, sensitive and specific staining of collagens plated in microtiter wells was achieved. The range of detection was from 0.5 to 20 micrograms. Human collagen types I, III, IV, and V were tested and able to be detected by the method. The dye did not bind to acetylcholinesterase or elastin. It did bind to C1q to some extent but this is not surprising since the molecule contains some triple helical collagen-like structures. A comparison performed between this assay and a colorimetric assay for hydroxyproline using tissue culture supernatants gave similar results for both samples. Due to its simplicity and sensitivity this assay will be most useful in laboratories where large numbers of samples must be screened for collagen production.     

Collagen crosslinks: occurrence in basement membrane collagens

Basement membrane preparations from anterior lens capsule, Descemet's 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.