Altered ratio of collagen chains in bone of a patient with non-lethal osteogenesis imperfecta.

Bone from a patient with osteogenesis imperfecta contained type III collagen which was absent in control bone. The ratio of alpha 1(I)/alpha 2(I) in type I collagen of patient's bone was increased (2.9 vs. 2.3 +/- 0.2 in controls) and the ratio of dimers beta 11/beta 12/beta 22 was altered due to the increased beta 22 content. No abnormality was observed in collagen from the patient's skin. The altered composition of collagen in bone, but the normal composition in skin suggests that the disease in the patient is due to impaired regulation of the synthesis of collagens in bone, rather than by a mutation in one of the two type I collagen genes. Unlike in skin, all the type III collagen in patient's bone was pepsin-soluble indicating an inability of the bone to incorporate type III collagen into mature highly cross-linked extracellular matrix.

[Structural characteristics of collagens from the skin and rib cartilage of patients with Ehlers-Danlos syndrome type II]. / Strukturnye kharakteristiki kollagenov kozhi i rebernogo khriashcha bol'nykh s sindromom Elersa-Danlo II tipa]

Collagens were analyzed in skin and rib cartilage of 9 patients with Ehlers-Danlos syndrome of the II type. Electrophoresis and CNBr-peptide mapping showed that extended inserts and deletions as well as rough impairments of post-translation processing were not detected in collagens of the I, II and III types from these patients. In the patients with Ehlers-Danlos syndrome of the II type distinct increase was observed both in the total ratio of collagens III/I (P = 0.95) and in the ratio of intact collagens III/I free of cross-links. A decrease in content of dimers beta 11 and beta 12 was found in two patients. The data obtained suggest that the Ehlers-Danlos syndrome of the II type involved deteriorations in the structure of collagens I responsible for decrease in stability and sometimes for impairments in cross-link formation. Increase in content of collagen II fraction, predisposed to proteolytic hydrolysis of terminal sites, as well as elevated sensitivity of collagen II to pepsin hydrolysis were found in collagens of rib cartilage from patients with the syndrome and with funnel chest deformation. This suggests the lowered stability of collagen II from rib cartilage in funnel chest deformation.

[Changes in the structure of type I collagen and cross-links between type I and type III collagen chains in a patient with funnel chest]. / Narushenie struktury kollagena I tipa i nalichie sshivok mezhdu tsepiami kollagenov I i III tipa u bol'nogo s izolirovannoi formoi kilevidnoi deformatsii grudnoi kletki.

Skin and rib cartilage collagens were studied in patient 1.I.K. with isolated form of pigeon chest as well as in a group of children without any impairments of connective tissue. Distinct decrease in stability of collagen I, an increase in the ration of alpha 1 (I)/alpha 2(I) chains and impairment in formation of beta 12 dimers were detected in the patient with pigeon chest. In the patient skin total ratio between collagens I and III, calculated from a content of BrCN-peptides, was similar to normal level, whereas the proportion was markedly increased between intact molecules of collagens III and I free of cross-links, which was calculated from the ratio of alpha 1(III)/alpha 2(I) chains. Presence of cross-links between alpha 1 (III) and alpha 2 (I) chains as well as between alpha 1 (III) and alpha 2 (I) chains was detected after peptide mapping of polypeptides arranged in the region of beta 11 and beta 12 dimers. All the collagen I preparation, extracted from skin of the patient 1.I.K., contained molecules with unstabilized N-terminal sites. These results suggest that mutation occurred in the N-terminal region of alpha 1(I) chain. Analysis of collagen from the patient 1.I.K. rib cartilage demonstrated a slight decrease in total stability of collagen II as well as elevated concentration of collagen II molecules containing unstabilized N-terminals. Mechanisms responsible for formation of cross-links between polypeptide chains of collagens I and III detected in human skin are discussed.

The absence of type II collagen and changes in proteoglycan structure of hyaline cartilage in a case of Langer-Saldino achondrogenesis.

Structural analysis of hyaline cartilage extracellular matrix components from the ribs and knee joint of a stillborn female with type II achondrogenesis was carried out. The absence of type II collagen, a decrease in the amount of proteoglycans (PG), and structural changes in PG, namely, increased electrophoretic mobility of PG, lower relative content of chondroitin 4-sulfate (Ch4-S), lower molecular weight and decreased total chondroitin sulfate (ChS) sulfation, were detected. Increased amounts of type I and type III collagens, atypical for hyaline cartilage, were revealed. Among the link proteins (LPs), a large protein with a mol. wt. of 48 kDa was predominant. Molecular and cellular mechanisms of the pathogenesis of achondrogenesis ("chondrogenesis imperfecta") are discussed. The data obtained suggest that the primary defect in type II achondrogenesis involves ChS or type II collagen synthesis.

Modified method for peptide mapping of collagen chains using cyanogen bromide-cleavage of protein within polyacrylamide gels.

A highly efficient method for cyanogen bromide (CNBr)-mapping of collagen peptides is described. This method was developed based on polypeptide cleavage by CNBr within gel slices according to Barsh et al. [1981) Collagen Relat. Res. 1, 543-548). The proposed method has the following advantages: (i) Analysis of both radiolabeled and unlabeled collagens is possible; (ii) CNBr-cleavage of polypeptides is performed in gel pieces which contain individual bands; (iii) The peptide losses are minimized, offering a more complete analysis of collagens including the low molecular weight CNBr-peptides.

[Heterogeneity of collagen molecules types I and II according to their resistance to proteolysis]. / Geterogennost' molekul kollagenov I i II tipov po ustoichivosti k proteolizu.

Study of the effects of pepsin treatment on soluble collagens type I of the skin and collagens type II of the costal cartilage of healthy subjects revealed the presence of two classes of molecules differing in the stability of their three-helical structure. In collagen molecules possessing a low stability (their number may amount to 20-30%) within the temperature range of 4-30 degrees C pepsin causes a split-off of N-terminal sites with the formation of short chains, i.e., alpha 1(I), alpha 2(II), and alpha 1(II), whereas at higher temperatures (33 degrees C for collagens type I and 37 degrees C for collagens type II) a complete degradation of these molecules takes place. It was found that collagens types I and II molecules contain a high number of three-helical sites with a high susceptibility to pepsin. The putative functional role of structural heterogeneity of collagen molecules is discussed.

[Cyanogen bromide peptide mapping of collagen polypeptides after separation using polyacrylamide gel electrophoresis and detection using staining or autoradiography]. / Bromtsianovoe peptidnoe kartirovanie kollagenovykh polipeptidov posle razdeleniia élektroforezom v poliakrilamidnom gele i detektsii okrashivaniem ili radioavtografiei.

Influence of various procedures for detection of collagen chains after electrophoresis in polyacrylamide gel on efficiency of their subsequent electroelution from the gel slices into gel of the second direction was studied. Staining of the gels with Coumassy R-250 under mild conditions or radioautography of the gels dried after electrophoresis decreased only slightly the electroelution efficiency as compared with the untreated gels. This observation suggests that direct procedures, which simplified distinctly the experiments, might be used for detection of the polypeptides in initial gel when peptide mapping was carried out using degradation of collagens by means of cyanogen bromide immediately in the gel slices. The procedures were developed, which enabled to perform mapping of collagen individual chains both labelled with 14C-amino acids and unlabelled fractions after their electrophoresis in polyacrylamide gel and the direct detection of polypeptides.

[Abnormal structure of type II collagen in a patients with funnel chest]. / Anomaliia struktury kollagena II tipa u bol'nogo s voronkoobraznoi deformatsiei grudnoi kletki.

The electrophoretical analysis and CNBr-peptide mapping of the collagens, isolated from the costal cartilage of 30 patients with non-classified and syndromal forms of pex excavatum (funnel chest) (27 patients) and pex carinatum (3 patients) was carried out. In case of one patient with the nonclassified form of funnel chest the electrophoretical mobility of CB 9.7-peptide was found to be decreased. The electrophoretical mobilities of other peptides are not markedly changed. The data obtained allow one to suggest the mutation causing the defect in the region about 160 amino acid residues distant from the C-end of alpha 1 (II) chain of type II collagen of the patient.

[Optimization of the method of cyanogen bromide mapping of polypeptides separated by polyacrylamide gel electrophoresis]. / Optimizatsiia metoda rasshchepleniia bromtsianom polipeptidov, razdelennykh élektroforezom v poliakrilamidnom gele.

Two highly efficient methods of CNBr-peptide mapping of polypeptides divided by polyacrylamide gel electrophoresis are described. The first is elaborated on the basis of peptide mapping of collagen proposed by G. Barsh et al. The following three modifications diminish wasting the material essential for the method. 1. CNBr treatment takes place in the absence of CNBr solution outside the gel, excluding the peptides elution from the gel fragments in the process of mapping. 2. After CNBr treatment the solution of CNBr is substituted by the samples buffer before electrophoresis by means of drying and subsequent addition of minimal volumes of the buffer. The latter procedures substitute the gel washing out by the buffer solution. 3. The step of washing the gel fragments by the 70% strong solution of formic acid before CNBr treatment is excluded. The second method of CNBr-peptide mapping is notable for extracting peptides from the gel fragments in the process of CNBr-treatment and permits obtaining of the high quality peptide electrophoregrams.

[Various characteristics of the structure and synthesis of procollagens produced by cultured skin fibroblasts from patients with Danlos-Ehlers syndrome type I]. / Nekotorye kharakteristiki struktury i sinteza prokollagenov, produtsiruemykh kul'turami fibroblastov kozhi bol'nykh s sintromom Elersa-Danlo I tipa.

The electrophoretic mobilities of the collagen and procollagen type I and III chains synthesized by the fibroblasts isolated from patients with type I Ehlers-Danlos syndrome as well as a set of peptides obtained by splitting of pro alpha 1(I) and pro alpha 2(I) type I procollagens by cyanbromide are not different from the normal ones. The fact demonstrates the absence of long insertions or deletions, or the sufficient defects in intracellular chain modifications. The changes were also nor registered for the ratio of type I and III collagens from the digested by pepsin preparations of protein accumulating in the culture media of the cultured skin fibroblasts from patients. The studied strains of cultured fibroblasts from patients suffering the Ehlers-Danlos syndrome have the trend to increased accumulation of partially processed chains of proc alpha 1(I) and proc alpha 2(I) type I procollagen and to the increased ratio of pro alpha 1(I) to pro alpha 2(I).