Structure of cDNA clones coding for human type II procollagen. The alpha 1(II) chain is more similar to the alpha 1(I) chain than two other alpha chains of fibrillar collagens.

Overlapping cDNA clones were isolated for human type II procollagen. Nucleotide sequencing of the clones provided over 2.5 kb of new coding sequences for the human pro alpha 1(II) gene and the first complete amino acid sequence of type II procollagen from any species. Comparison with published data for cDNA clones covering the entire lengths of the human type I and type III procollagens made it possible to compare in detail the coding sequences and primary structures of the three most abundant human fibrillar collagens. The results indicated that the marked preference in the third base codons for glycine, proline and alanine previously seen in other fibrillar collagens was maintained in type II procollagen. The domains of the pro alpha 1(II) chain are about the same size as the same domains of the pro alpha chains of type I and type III procollagens. However, the major triple-helical domain is 15 amino acid residues less than the triple-helical domain of type III procollagen. Comparison of hydropathy profiles indicated that the alpha chain domain of type II procollagen is more similar to the alpha chain domain of the pro alpha 1(I) chain than to the pro alpha 2(I) chain or the pro alpha 1(III) chain. The results therefore suggest that selective pressure in the evolution of the pro alpha 1(II) and pro alpha 1(I) genes is more similar than the selective pressure in the evolution of the pro alpha 2(I) and pro alpha 1(III) genes.     

COOH-terminal propeptides of the major human procollagens. Structural, functional and genetic comparisons

The sequences of the carboxy-terminal extensions (COOH-propeptides) of at least one chain of all of the major human procollagens have only recently been deduced, and include those of the interstitial (alpha 1(I), alpha 2(I), alpha 1(II), alpha 1(III)), basement membrane (alpha 1(IV)) and pericellular (alpha 2(V)) procollagens. Comparisons of DNA and protein sequences, corresponding to these COOH-propeptides domains, established the early divergence of the basement membrane alpha 1(IV) COOH-propeptide from the corresponding sequences of the interstitial and pericellular procollagens. The latter are relatively highly conserved and share 58% primary peptide sequence similarities, whereas sequence similarities relative to alpha 1(IV) are limited. Hydropathy profiles and secondary structure potentials further emphasize the clustering of conserved and variable regions among the interstitial and pericellular COOH-propeptides, and provided further evidence for significant structural differences between these sequences and the alpha 1(IV) COOH-propeptide. The most highly conserved sequences of the alpha 1(I), alpha 2(I), alpha 1(II), alpha 1(III) and alpha 2(V) COOH-propeptides include regions surrounding the carbohydrate attachment site, cysteine-containing regions and the COOH-terminal sequences. Cysteinyl, tyrosyl and tryptophanyl residues were found to be highly conserved as were most charged residues. Localization of variable regions, in general, occurs within hydrophilic sequences with high beta-turn potentials that are proximal to intron/exon splice junctions. The most variable sequences are associated with the telopeptides and adjoining NH2-terminal portions of the COOH-propeptides as demonstrated by predictive secondary structure analyses. These results, combined with similar analyses of abnormal alpha 2(I) COOH-propeptide (osteogenesis imperfecta) permitted the identification of subsequences that are likely to be a prerequisite for COOH-propeptide functions, namely procollagen chain recognition and nucleation sites for triple helix formation. These functions are also common to the alpha 1(IV) COOH-propeptide; however, the lack of cleavage of this region and its additional postulated structural role in extracellular matrix interactions likely account for its divergent primary and secondary structure.     

Mutations that alter the primary structure of type I procollagen have long-range effects on its cleavage by procollagen N-proteinase

Type I/II procollagen N-proteinase was partially purified from chick embryos and used to examine the rate of cleavage of a series of purified type I procollagens synthesized by fibroblasts from probands with heritable disorders of connective tissue. The rate of cleavage was normal with procollagen from a proband with osteogenesis imperfecta that was overmodified by posttranslational enzymes. Therefore, posttranslational overmodification of the protein does not in itself alter the rate of cleavage under the conditions of the assay employed. Cleavage of the procollagen, however, was altered in several procollagens with known mutations in primary structure. Two of the procollagens had in-frame deletions of 18 amino acids encoded by exons 11 and 33 of the pro alpha 2(I) gene. In both procollagens, both the pro alpha 1(I) and the pro alpha 2(I) chains were totally resistant to cleavage. With a procollagen in which glycine-907 of the alpha 2(I) chain domain was substituted with aspartate, both pro alpha chains were cleaved but at a markedly decreased rate. The results, therefore, establish that mutations that alter the primary structure of the pro alpha chains of procollagen at sites far removed from the N-proteinase cleavage site can make the protein resistant to cleavage by the enzyme. The long-range effects of in-frame deletions or other changes in amino acid sequence are probably explained by their disruption of the hairpin structure that is formed by each of the three pro alpha chains in the region containing the cleavage site and that is essential for cleavage of the procollagen molecule by N-proteinase.     

Type I procollagens containing substitutions of aspartate, arginine, and cysteine for glycine in the pro alpha 1 (I) chain are cleaved slowly by N-proteinase, but only the cysteine substitution introduces a kink in the molecule.

Type I procollagen was purified from the medium of dermal fibroblasts cultured from four individuals with osteogenesis imperfecta (OI) type II who had mutations in the COL1A1 gene of type I procollagen. The procollagens were mixtures of normal molecules and molecules that contained substitutions of aspartate for glycine 97, arginine for glycine 550, cysteine for glycine 718, and aspartate for glycine 883 in one or both of the alpha 1 (I) chains of the molecule. The procollagens were cleaved more slowly than control type I procollagen by procollagen N-proteinase. Double-reciprocal plots of initial relative velocities and initial substrate concentrations indicated that the OI procollagens were all cleaved slowly by N-proteinase because of decreased Vmax, rather than increased Km. This suggested that slow cleavage of the OI procollagens by N-proteinase was the result of slow conversion of the N-proteinase-procollagen complex. Further experiments showed that the vertebrate collagenase A fragment of the aspartate for glycine alpha 1(I) 883 OI procollagen that contained the N-proteinase cleavage site but not the site of the substitution was also cleaved more slowly by N-proteinase than the normal vertebrate collagenase A fragments in the samples. These data show, for the first time, that an altered triple-helical structure is propagated from the site of a substitution of a bulky residue for glycine to the amino-terminal end of the procollagen molecule and disrupts the conformation of the N-proteinase cleavage site. Rotary shadowing electron microscopy of molecules in the preparation of cysteine for glycine alpha 1(I)-718 showed the presence of a kink in approximately 5% of a population of molecules in which 60% were abnormal and 20% contained a disulfide bond. In contrast, procollagens containing aspartate and arginine for glycine were indistinguishable by rotary shadowing electron microscopy from those in control samples. The results here confirm previous suggestions that substitution of cysteine for glycine in the alpha 1(I) chain of type I collagen can introduce a kink near the site of the substitution. However, the presence of a kink is not a prerequisite for delayed cleavage of abnormal procollagens by N-proteinase.     

Different features of the MHC class I heterodimer have evolved at different rates. Chicken B-F and beta 2-microglobulin sequences reveal invariant surface residues.

Chicken beta 2-microglobulin (beta 2m) and class I (B-F19 alpha chain) cDNA clones were isolated and the sequences compared to those of B-F Ag isolated from chicken E. These clones represent the major expressed class I molecules on E, with B-F alpha size variants evidently due to alternative use of small exons in the cytoplasmic region. The cDNA sequences were compared to turkey beta 2m, the apparent allele B-F12 alpha and other vertebrate homologs, using the 2.6 A structure of the human HLA-A2 molecule as a model. Both chicken alpha 1 and alpha 2 domains resemble mammalian classical class I molecules and the MHC-encoded nonclassical molecules more than CD1 or the class I-like FcR. In contrast, the chicken alpha 3 domain is equally homologous to all alpha 3 domains, to beta 2m and to class II beta 2 domains. For each pair of extracellular domains (alpha 1 vs alpha 2, alpha 3 vs beta 2m), the level of sequence homology between mammalian and avian molecules is quite different. This suggests that the structurally homologous domains have been under different selective pressures during evolution. There is a very strong G + C bias in alpha 3 and beta 2m, leading to an overall change in amino acid composition in B-F compared to class I molecules from other taxa. Many of the surface residues are quite diverged, particularly in alpha 3 and beta 2m. There are fewer changes in intra- and interdomain contact sites. Some residues with important functions are invariant, including seven residues that bind the ends of the peptide, two residues that bind CD8, and three residues that are phosphorylated. The positions of the allelic residues are conserved. There are other patches of invariant residues on alpha 1, alpha 2, and beta 2m; these might bind TCR or other molecules involved in class I function.     

Amino-terminal propeptide of human pro-alpha 2(V) collagen conforms to the structural criteria of a fibrillar procollagen molecule

We have determined the nucleotide sequence of a cDNA clone encoding the amino-terminal portion of human alpha 2(V) procollagen and found that the structure of the 186-residue amino-terminal propeptide closely resembles those of the fibril-forming procollagens. Juxtaposed to a 26-residue leader peptide, pro-alpha 2(V) exhibits a characteristic cysteine-rich globular region followed by 24 Gly-X-Y repeats which are interrupted by two short non-collagenous sequences. Upon closer examination, each of these two sequences was noted to display structural motifs characteristic of either pro-alpha 1(I) and pro-alpha 1(III) collagens or pro-alpha 1(II) collagen, respectively. Finally, within the amino-terminal telopeptide, a putative amino-terminal proteinase cleavage site, Ala-Gln, was identified. This latter finding strongly suggests that the alpha 2(V) amino-terminal propeptide can be potentially processed and thus leaves unresolved the issue pertaining to the nature of the collagenase-resistant sequence that is retained by mature type V collagen molecules.     

Immune interferon suppresses levels of procollagen mRNA and type II collagen synthesis in cultured human articular and costal chondrocytes

Cultured human articular and costal chondrocytes were used as a model system to examine the effects of recombinant gamma-interferon (IFN-gamma) on synthesis of procollagens, the steady state levels of types I and II procollagen mRNAs, and the expression of major histocompatibility complex class II (Ia-like) antigens on the cell surface. Adult articular chondrocytes synthesized mainly type II collagen during weeks 1-3 of primary culture, whereas types I and III collagens were also produced after longer incubation and predominated after the first subculture. Juvenile costal chondrocytes synthesized no detectable alpha 2(I) collagen chains until after week 1 of primary culture; type II collagen was the predominant species even after weeks of culture. The relative amounts of types I and II collagens synthesized were reflected in the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. In articular chondrocytes, the levels of alpha 1(I) procollagen mRNA were disproportionately low (alpha 1(I)/alpha 2(I) less than 1.0) compared with costal chondrocytes (alpha 1 (I)/alpha 2(I) approximately 2). Recombinant IFN-gamma (0.1-100 units/ml) inhibited synthesis of type II as well as types I and III collagens associated with suppression of the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. IFN-gamma suppressed the levels of alpha 1(I) and alpha 1(II) procollagen mRNAs to a greater extent than alpha 2(I) procollagen mRNA in articular but not in costal chondrocytes. Human leukocyte interferon (IFN-alpha) at 1000 units/ml suppressed collagen synthesis and procollagen mRNA levels to a similar extent as IFN-gamma at 1.0 unit/ml. In addition, IFN-gamma but not IFN-alpha induced the expression of HLA-DR antigens on intact cells. The lymphokine IFN-gamma could, therefore, have a role in suppressing cartilage matrix synthesis in vivo under conditions in which the chondrocytes are in proximity to T lymphocytes and their products.     

Location of the 11 bp exon in the chicken pro alpha 2(I) collagen gene.

During the fine structural analysis of the 5' end of the 38 kb chicken pro alpha 2(I) collagen gene, we failed to locate an exon, only 11 bp in size, which had been predicted from the DNA sequence analysis of a cDNA clone complementary to the 5' end of the pro alpha 2(I) collagen mRNA (1). We know report the location of this 11 bp exon, exon 2, at the 5' end of a 180 bp Pst I fragment, 1900 bp 3' to exon 1 and 600 bp 5' to exon 3. Its sequence, ATGTGAGTGAG, is highly unusual in that it contains two overlapping consensus donor splice sequences. Moreover, it is flanked by two overlapping donor splice sequences but only one of the four splice sequences is actually spliced (1). The first half of intron 1 also has an unusual sequence: it is 68% GC, contains 88 CpG dinucleotides and 11 Hpa II sites. The second half is more like other intron sequences in the collagen gene with a GC content of 41%, 19 CpG, and no Hpa II sites. However it contains two sequences with 7 and 9 bp homology to the 14 bp SV40 enhancer core sequence. It is suggested that some part of intron 1 may be involved in regulation.     

Bone morphogenetic protein 1 is an extracellular processing enzyme of the laminin 5 gamma 2 chain

Epithelial cells maintained in culture medium containing low calcium proteolytically process laminin 5 (alpha3beta3gamma2) within the alpha3 and gamma2 chains (). Experiments were designed to identify the enzyme(s) responsible for the laminin 5 processing and the sites of proteolytic cleavage. To characterize the nature of laminin 5 processing, we determined the N-terminal amino acid sequences of the proteolytic fragments produced by the processing events. The results indicate that the first alpha3 chain cleavage (200-l65 kDa alpha3) occurs within subdomain G4 of the G domain. The second cleavage (l65-l45 kDa alpha3) occurs within the lIla domain, 11 residues N-terminal to the start of domain II. The gamma chain is cleaved within the second epidermal growth factor-like repeat of domain Ill. The sequence cleaved within the gamma2 chain matches the consensus sequence for the cleavage of type I, II, and III procollagens by bone morphogenetic protein-1 (BMP-1), also known as type I procollagen C-proteinase (). Recombinant BMP-1 cleaves gamma2 in vitro, both within intact laminin 5 and at the predicted site of a recombinant gamma2 short arm. alpha3 is also cleaved by BMP-1 in vitro, but the cleavage site is yet to be determined. These results show the laminin alpha3 and gamma2 chains to be substrates for BMP-1 in vitro. We speculate that gamma2 cleavage is required for formation of the laminin 5-6 complex and that this complex is directly involved in assembly of the interhemidesmosomal basement membrane. This further suggests that BMP-1 activity facilitates basement membrane assembly, but not hemidesmosome assembly, in the laminin 5-rich dermal-epidermal junction basement membrane in vivo.     

Pancreatic beta cell-specific transcription of the pdx-1 gene. The role of conserved upstream control regions and their hepatic nuclear factor 3beta sites

To identify potential transactivators of pdx-1, we sequenced approximately 4.5 kilobases of the 5' promoter region of the human and chicken homologs, assuming that sequences conserved with the mouse gene would contain critical cis-regulatory elements. The sequences associated with hypersensitive site 1 (HSS1) represented the principal area of homology within which three conserved subdomains were apparent: area I (-2694 to -2561 base pairs (bp)), area II (-2139 to -1958 bp), and area III (-1879 to -1799 bp). The identities between the mouse and chicken/human genes are very high, ranging from 78 to 89%, although only areas I and III are present within this region in chicken. Pancreatic beta cell-selective expression was shown to be controlled by mouse and human area I or area II, but not area III, from an analysis of pdx-1-driven reporter activity in transfected beta- and non-beta cells. Mutational and functional analyses of conserved hepatic nuclear factor 3 (HNF3)-like sites located within area I and area II demonstrated that activation by these regions was mediated by HNF3beta. To determine if a similar regulatory relationship might exist within the context of the endogenous gene, pdx-1 expression was measured in embryonic stem cells in which one or both alleles of HNF3beta were inactivated. pdx-1 mRNA levels induced upon differentiation to embryoid bodies were down-regulated in homozygous null HNF3beta cells. Together, these results suggest that the conserved sequences represented by areas I and II define the binding sites for factors such as HNF3beta, which control islet beta cell-selective expression of the pdx-1 gene.     

Association of MHC class I and class II gene polymorphisms with vaccine or challenge response to Salmonella enteritidis in young chicks

Salmonella enteritidis (SE) is a worldwide source of salmonellosis in humans, caused mainly by consumption of contaminated poultry products. The major histocompatibility complex (MHC) class I and class II molecules, which function as antigen-presentation structures, are involved in both macrophage and dendritic cell immune response to Salmonella and may, therefore, play an important role in host resistance to infections. The chicken MHC class I and class II were investigated as candidate genes for immune response to SE. We characterized the complete MHC class I cDNA sequences for an outbred broiler line and four diverse highly inbred lines: two MHC-congenic Leghorn (G-B2 and G-B1), one Egyptian Fayoumi (M15.2), and one Spanish (Sp21.1), to define the allelic sequences within these lines, so that we might study the association between particular MHC polymorphisms and response to SE. The F1 offspring of outbred broiler sires crossed with three inbred lines (G-B1, G-B2, and Fayoumi) were evaluated as young chicks for either bacterial load in spleen and cecum after pathogenic SE inoculation or antibody level after SE vaccination. Alleles defined by a Lys(148)-->Met(148) polymorphism in the MHC class I alpha(2) domain were associated with spleen bacterial load after SE challenge. These results suggest that particular MHC haplotypes may contribute to control of responses to SE, and that particular polymorphisms may serve as markers for genetic resistance to SE in the chicken.     

Analysis of cDNA and genomic clones coding for the pro alpha 1 chain of calf type II collagen.

A bovine cDNA library constructed from fetal cartilage RNA was screened with a pro alpha 1(II) collagen specific chicken cDNA. A recombinant clone (Bc 7), with an insert of 1 kb, was identified and shown to contain sequences exhibiting 85% homology with the chicken pro alpha 1(II) collagen C-propeptide. Interspecies comparison strongly suggested that one potential glycosylation site present in the avian C-propeptide is not utilized, since this site is absent in the bovine chain. In addition, two overlapping genomic clones (Pal 3 and Pal 4) were isolated and partially characterized. These clones span 23 kb of DNA and contain approximately 17 kb of the pro alpha 1(II) calf gene. Sequencing of exon 1 has determined the length of the 3' untranslated region and the exact location of the polyadenylation attachment site.     

Purification, characterization, and distribution of enolase isozymes in chicken

Chicken brain enolase was found to show multiple forms (I, II and III) separable by DEAE-cellulose column chromatography, whereas enolase from chicken skeletal muscle showed a single form. Brain enolase I, enolase III and muscle enolase were purified to electrophoretic homogeneity. These three isozymes were dimeric enzymes, each being composed of two identical subunits, alpha, gamma and beta, having molecular weight of 51,000 +/- 600, 52,000 +/- 550 and 51,500 +/- 650, respectively, as determined by SDS-polyacrylamide gel electrophoresis analysis. Brain enolases I, II and III and muscle enolase had similar catalytic parameters, including almost the same Km values and pH optima. Specific antibodies against brain enolase I, enolase III and muscle enolase, raised in rabbit, showed no cross-reactivity with each other. Antibodies for brain enolases I and III also reacted with brain enolase II, indicating that brain enolase II was the hybrid form (alpha gamma) of brain enolases I (alpha alpha) and III (gamma gamma). Enolases from chicken liver, kidney and heart reacted with the antisera for brain enolase I, but not with those for brain enolase III or muscle enolase. Developmental changes in enolase isozyme distribution were observed in chicken brain and skeletal muscle. In brain, the alpha gamma and gamma gamma forms were not detected in the early embryonic stage and increased gradually during the development of the brain, whereas the alpha alpha form existed at an almost constant level during development. In skeletal muscle, complete switching from alpha alpha enolase to beta beta was observed during the period around hatching.     

Inhibition of bone morphogenetic protein 1 by native and altered forms of alpha2-macroglobulin

The four mammalian bone morphogenetic protein 1 (BMP1)-like proteinases act to proteolytically convert procollagens to the major fibrous components of the extracellular matrix. They also activate lysyl oxidase, an enzyme necessary to the covalent cross-linking that gives collagen fibrils much of their tensile strength. Thus, these four proteinases are attractive targets for interventions designed to limit the excess formation of fibrous collagenous matrix that characterizes fibrosis. Although it has previously been reported that the serum protein alpha(2)-macroglobulin is unable to inhibit the astacin-like proteinases meprin alpha and meprin beta, we herein demonstrate alpha(2)-macroglobulin to be a potent inhibitor of the similar BMP1-like proteinases. BMP1 is shown to cleave the alpha(2)-macroglobulin "bait" region, at a single specific site, which resembles the sites at which BMP1-like proteinases cleave the C-propeptides of procollagens I-III. alpha(2)-Macroglobulin is an irreversible inhibitor that is shown to bind bone morphogenetic protein 1 in a covalent complex. It is also demonstrated that genetically modified alpha(2)-macroglobulin, in which the native bait region is replaced by sequences flanking the probiglycan BMP1 cleavage site, is enhanced approximately 24-fold in its ability to inhibit BMP1, and is capable of inhibiting the biosynthetic processing of procollagen I by cells. These findings suggest possible therapeutic interventions involving ectopic expression of modified versions of alpha(2)-macroglobulin in the treatment of fibrotic conditions.     

Identification of genomic DNA coding for chicken type II procollagen

A segment of the type II procollagen gene has been isolated by screening a lambda Charon 4A library containing fragments of chicken genomic DNA. The specific clone, LgCOL(II), was selected by hybridization using overlapping inserts from two cDNA clones which are specific for a cartilage procollagen (Vuorio, E., Sandell, L., Kravis, D., Sheffield, V. C., Vuorio, T., Dorfman, A., and Upholt, W. B. (1982) Nucleic Acids Res. 10, 1175-1192). DNA sequence analysis of LgCOL(II) in the COOH-telopeptide region of the protein, shows conclusively that this DNA corresponds to the chicken type II procollagen gene. Hybridization of cDNA probes to restriction fragment gel blots together with DNA sequence analysis have established the orientation and position of the procollagen gene within the lambda Charon 4A vector and indicate that LgCOL(II) contains approximately 6 kilobase pairs of the type II procollagen gene plus additional DNA flanking the 3' end of the gene. DNA sequence analysis shows directly that LgCOL(II) contains DNA sequences identical with those in the cDNA clones. The portion of the gene from amino acid 578 of the triple helical region to the COOH-terminal end of the protein (approximately 700 amino acids) is contained within the clone, corresponding to approximately 50% of the amino acid coding sequence of the gene. This region of the chicken alpha 1 (type II) procollagen gene is encoded within a shorter segment of the chicken genome than is the corresponding region of the alpha 2(type I) procollagen gene.     

Isolation and partial characterization of the amino-terminal propeptide of type II procollagen from chick embryos

The amino-terminal propeptide from type II procollagen was isolated from organ cultures of sternal cartilages from 17-day-old chick embryos. The procedure provided the first isolation of the propeptide in amounts adequate for chemical characterization. The propeptide had an apparent molecular weight of 18000 as estimated by gel electrophoresis in sodium dodecyl sulfate. It contained a collagen-like domain as demonstrated by its amino acid composition, circular dichroism spectrum, and susceptibility to bacterial collagenase. One residue of hydroxylysine was present, the first time this amino acid has been detected in a propeptide. The peptide contained no methionine and only two residues of half-cystine. Antibodies were prepared to the propeptide and were used to establish its identity. The antibodies precipitated type II procollagen but did not precipitate type II procollagen from which the amino and carboxy propeptides were removed with pepsin. Also, they did not precipitate the carboxy propeptide of type II procollagen. The data demonstrated th at the type II amino propeptide was similar to the amino propeptides of type I and type III procollagens in that it contained a collagen-like domain. It differed, however, in that it lacked a globular domain as large as the globular domain of 77-86 residues found at the amino-terminal ends of the pro alpha 1 chains of type I and type III procollagens.     

cDNA cloning of the chicken branched-chain alpha-keto acid dehydrogenase complex. Chicken-specific residues of the acyltransferase affect the overall activity and the interaction with the dehydrogenase.

Branched-chain alpha-keto acid dehydrogenase complex is a macromolecule comprising three catalytic components: a dehydrogenase (E1) with alpha(2)beta(2) structure, an acyltransferase (E2) and a dihydrolipoamide dehydrogenase (E3). In the mammalian complex, the E2 component with 24 identical subunits forms a structural core, to which multiple copies of E1 and E3 bind noncovalently. We isolated cDNA clones encoding E1 alpha, E1 beta and E2 subunits from a chicken-liver cDNA library and performed nucleotide sequencing. Amino-acid sequences deduced from the nucleotide sequences revealed that chicken E1 alpha and E1 beta chains had substantially homologous sequences with the corresponding mammalian polypeptides, except for the N-terminus. Chicken E2 conserved three functional domains, a lipoyl-bearing domain, an E1/E3 binding domain and an inner-core domain, but contrasted strongly with mammalian E2 in respect of containing 11 additional residues in two interdomain linkers: nine sequential residues in one linker and two residues in the other. Replacement of many residues was also observed in the chicken linkers. When E2 activity for catalyzing the overall reaction was measured by activity reconstitution in combination with E1 and E3, chicken E2 was markedly less effective than mammalian E2. The capability of chicken E2 for binding E1 was also reduced when determined by the binding assay using sucrose density gradient centrifugation. Chicken E1 was functionally as well as structurally indistinguishable from mammalian E1. Thus the reduced catalytic activity of chicken E2 must arise from its reduced E1-binding capacity, which results from the characteristic structure of interdomain linkers in chicken E2.     

Various characteristics of the structure and synthesis of procollagens produced by cultured skin fibroblasts from patients with Danlos-Ehlers syndrome type I

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).