Assembly and processing of procollagen type III in chick embryo blood vessels

The processing of [3H]proline-labeled procollagen III in excised chick embryo blood vessels was found to differ significantly from that of procollagen I in the same tissue. While first the amino propeptides and then the carboxyl propeptides were fairly rapidly cleaved from procollagen I, only the carboxyl propeptides were split off procollagen III, leaving pN-collagen III. This intermediate, which is only slowly converted to collagen III by loss of amino propeptides, was characterized by its sedimentation properties, isolation of the amino propeptide, and reaction with purified antibodies that are specific against bovine amino propeptide III. It is interchain disulfide-linked, both through the amino propeptide and the carboxyl ends of the collagen chains. The conversion of procollagen III to pN-collagen III either in blood vessels, or after isolation by a carboxyl procollagen peptidase obtained from chick tendon fibroblast cultures, is inhibited by 50 mM arginine. Underhydroxylated procollagen III was isolated from blood vessels treated with alpha, alpha'-dipyridyl. Its amino propeptides reacted with the above antibodies but were not linked to each other. In contrast, its carboxyl propeptides were interchain disulfide-bridged, supporting previous suggestions that the carboxyl propeptides play a role in the assembly of procollagen trimer.     

Complete primary structure of the human alpha 2 type V procollagen COOH-terminal propeptide

Recently we presented the partial covalent structure of a type V collagen chain. Analysis of amino acids 796-1020 in the human alpha 2(V) Gly-X-Y region showed strong conservation of charged positions with the interstitial collagens but also revealed substitutions unique to type V. To gain more information about this procollagen and primarily to resolve the ambiguous nature of the 3' noncollagenous propeptide, we sequenced several cDNA clones coding for amino acids adjacent to the carboxyl end of the alpha chain. Here we report the complete primary structure of the alpha 2(V) COOH-terminal propeptide. In general, the latter sequence (270 residues) bears a greater degree of similarity to those of the interstitial rather than the basement membrane procollagens. Compared to the interstitial procollagens, however, more divergence has occurred in alpha 2(V) surrounding the conserved N-asparaginyl-linked carbohydrate attachment site at residues 171-173, and alpha 2(V) possesses an additional potential glycosylation site (Asn-Lys-Thr) located in a hypervariable region near the NH2 terminus. Although certainly premature to form any rigid hypothesis, a pattern emerges that may be characteristic of alpha 2 versus alpha 1 chains. Both the alpha 2(I) and alpha 2(V) telopeptides are devoid of a lysine, which in alpha 1 chains forms an interchain cross-link with residue 87 of the collagenous region. Also in contrast to the interstitial alpha 1 carboxyl propeptides is the absence in alpha 2(I) and alpha 2(V) of a cysteine that probably participates in an interchain disulfide bond. Therefore, one can speculate that those alpha 2 chains, represented only once in procollagen trimers, may not be under the same selective pressure as alpha 1 chains to maintain certain residues responsible for stabilizing the triple helical molecules.     

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.     

Interchain disulfide bond formation in types I and II procollagen. Evidence for a protein disulfide isomerase catalyzing bond formation

The assembly of reduced pro-alpha chains of type I and type II procollagen into the native triple-helical molecule was examined in vitro in the presence and absence of pure protein disulfide isomerase. The data clearly indicates that protein disulfide isomerase is able to accelerate the formation of native interchain disulfide bonds in these procollagens. It takes about 6 min after disulfide bonding before triple-helical molecules exist, while the time required to produce triple-helical type I procollagen in the presence of protein disulfide isomerase is 9.4 min and that for type II procollagen 17.2 min. These values agree with those obtained for type I and II procollagen in vivo suggesting that protein disulfide isomerase is also an enzyme catalyzing interchain disulfide bond formation in procollagen in vivo. The formation of native disulfide bonds can proceed without any enzyme catalysis but then requires the presence of reduced and oxidized glutathione. Bonding is rather slow in such a case, however, resulting in a delay in the formation of the triple helix.     

Biosynthesis of collagen

During the biosynthesis and assembly of collagen structures, disulfide links can serve several functions. During biosynthesis they successively stabilize intra-peptide folding and associations of three chains into one molecule. Studies on the refolding and reassociation of reduced and denatured carboxyl propeptides of procollagen I showed that successive interactions of folding and assembly are successively weaker. Disulfide bridges were reestablished within correctly refolded carboxyl propeptides. Rearrangements of disulfide bridges may occur during the processing of type V procollagen molecules as these collagens become incorporated into extracellular matrix. The basement membrane procollagen IV molecules become disulfide linked at each end into networks, and there are indications that further rearrangements of disulfide links may allow additional modulation.     

Folding of carboxyl domain and assembly of procollagen I

An early form of procollagen I was found in acetic acid extracts of radioactively labeled chick embryo skull bones. It resembled native procollagen I, but sedimented slightly faster, and its component chains were slightly underhydroxylated and were not disulfide-linked to each other, although its propeptides were internally disulfide-bonded. Pulse-chase experiments showed its conversion to disulfide-linked procollagen. As the same conversion occurred when proline hydroxylation was blocked by 2,2'-dipyridyl, we infer that the formation of this precursor from its component chains does not require collagen triple helix formation. We suggest that interaction between the folded carboxyl propeptides of individual pro-alpha (I) chains is an important step in the formation of this precursor and of procollagen I. Studies of the refolding and association of fully reduced and denatured carboxyl propeptides supported this concept. In the presence of glutathione the correct disulfide bonds could be reestablished, as judged by a mapping of some tryptic peptides. Individual carboxyl propeptides refolded first, and this occurred even in 2 M urea. Recognition between folded carboxyl propeptides occurred only when less than 0.5 M urea was present. The presence of the carboxyl telopeptides was important for trimeric reassembly. Individual propeptides also folded spontaneously during cell-free translation of pro-alpha (I) chains and were recognized by specific antibodies. We consider the role of carboxyl propeptides in the formation of procollagen I molecules and suggest a model of self-assembly, possibly facilitated by interactions with the luminal surface of the rough endoplasmic reticulum.     

Isolation and purification of human type I procollagen by adsorption to glass beads.

Procollagen from the culture medium of human foreskin fibroblasts is efficiently adsorbed on controlled-pore glass (CPG) beads. Elution of adsorbed protease(s), capable of procollagen degradation, is accomplished with 1 m phosphate. This allows subsequent purification steps to be accomplished without detectable degradation of the high-molecular-weight procollagen form which is subsequently eluted with 1 m Tris. Analysis of the Tris elution fraction from CPG beads by sodium dodecyl sulfate-agarose-polyacrylamide electrophoresis on 2% gels indicated that the majority of protein is types I and III procollagens and partially processed intermediates. Types I and III procollagens were separated by DEAE-cellulose chromatography, and presumptive undegraded type I procollagen was resolved from processed forms by molecular sieve chromatography in 1 m CaCl₂ on agarose beads. The high-molecular-weight type I human procollagen isolated by this method was found to contain both amino and carboxy-terminal propeptides. Two α1 and one α2 procollagen chains, disulfide bonded via the carboxy-terminal propeptides, are present per molecule. This procedure represents an efficient and relatively rapid method for preparing human procollagen in sufficient quantity for detailed chemical analysis.     

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.     

Biosynthesis and proteolytic processing of type XI collagen in embryonic chick sterna

The biosynthesis and proteolytic processing of type XI procollagen was examined using pulse-chase labelling of 17-day embryonic chick sterna in organ culture with [3H]proline. Products of biosynthesis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with and without prior reduction of disulfide bonds. Pro-alpha chains, intermediates, and matrix forms were identified by cyanogen bromide or Staphylococcus aureus V8 protease digestion. The results show that type XI pro-alpha chains assemble into trimeric molecules with interchain disulfide bonds. Proteolytic processing begins at least 40 min after the start of labeling which is later than that of type II procollagen (25 min). This first processing step involves the loss of the domain containing the interchain disulfide bonds which most likely is the carboxyl propeptide. In the case of the pro-alpha 3 chain, this generates the matrix form, m alpha 3, which retains its amino propeptide. For the pro-alpha 1 and pro-alpha 2 chains, this step generates intermediate forms, p alpha 1 and p alpha 2, which undergo a second proteolytic conversion to m alpha 1 and m alpha 2, and yet retain a pepsin-labile domain. The conversion of p alpha 2 to m alpha 2 is largely complete 2 h after labeling. p alpha 1 is converted to m alpha 1 very slowly and is 50% complete after 18 h of chase in organ culture. The apparent proteolytic processing within the amino propeptide, and the differential rate of processing between two chains in the same molecule are unusual and distinguish type XI from collagen types I, II, and III. It is possible that the extremely slow processing of p alpha 1 affects the formation of the heterotypic cartilage collagen fibrils and may be related to the function of type XI collagen.     

Identification of disulfide bonds in carboxy-terminal propeptides of human type I procollagen

Intra-chain and inter-chain disulfide bonds within the carboxy-terminal propeptides of human type I procollagen were studied using cyanogen bromide cleavage of the propeptides and electrophoresis on SDS-polyacrylamide/glycerol gels. The results could provide a better understanding of the assembly of pro alpha-chains into procollagen.     

Disulfide bonding as a determinant of the molecular composition of types I, II and III procollagen

Procollagen molecules have amino-terminal and carboxy-terminal propeptides at the respective ends of the collagenous triple helix. The carboxy-terminal propeptides enhance and direct the association of pro alpha-chains into procollagen molecules, but the mechanism of this registration function is still obscure. A hypothesis concerning the function of disulfide bonding in the assembly of types I, II and III procollagen is put forward here.     

Identification of the molecular recognition sequence which determines the type-specific assembly of procollagen.

A key question relating to procollagen biosynthesis is the way in which closely related procollagen chains discriminate between each other to assemble in a type-specific manner. Intracellular assembly of procollagen occurs via an initial interaction between the C-propeptides followed by vectorial propagation of the triple-helical domain in the C to N direction. Recognition signals within the C-propeptides must, therefore, determine the selective association of individual procollagen chains. We have used the pro alpha1 chain of type III procollagen [pro alpha1(III)] and the pro alpha2 chain of type I procollagen [pro alpha2(I)] as examples of procollagen chains that are either capable or incapable of self-assembly. When we exchanged the C-propeptides of the pro alpha1(III) chain and the pro alpha(I) chain we demonstrated that this domain is both necessary and sufficient to direct the assembly of homotrimers with correctly aligned triple-helices. To identify the sequences within this domain that determine selective association we constructed a series of chimeric procollagen chains in which we exchanged specific sequences from the pro alpha1(III) C-propeptide with the corresponding region within the pro alpha2(I) C-propeptide (and vice versa) and assayed for the ability of these molecules to form homotrimers. Using this approach we have identified a discontinuous sequence of 15 amino acids which directs procollagen self-association. By exchanging this sequence between different procollagen chains we can direct chain association and, potentially, assemble molecules with defined chain compositions.     

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.     

Sequential cleavage of type I procollagen by procollagen N-proteinase. An intermediate containing an uncleaved pro alpha 1(I) chain

The conversion of type I procollagen to type I collagen was studied by cleaving the protein with partically purified type I procollagen N-proteinase from chick embryos. Examination of the reaction products after incubation for varying times at 30 degrees C indicated that, during the initial stages of the reaction, pro alpha 1(I) and pro alpha 2(I) chains were cleaved at about the same rate. As a result, all the pro alpha 2(I) chains were converted to pC alpha 2(I) chains well before all the pro alpha 1 chains were cleaved. When the reaction products were examined by gel electrophoresis without reduction of interchain disulfide bonds, a distinct band of an intermediate was detected. The same intermediate was seen when the reaction was carried out at 35, 37, and 40 degrees C. The data established that over two-thirds of the type I procollagen was converted to the intermediate and that this intermediate was then slowly converted to the final product of pCcollagen. The kinetics for the reaction, however, did not fit a simple model for precursor-product relationship among substrate, intermediate, and product. Examination of the reaction products with a two-step gel procedure demonstrated that the intermediate consisted of three polypeptide chains in which the N propeptide was cleaved from one pro alpha 1 chain and one pro alpha 2(I) chain but the N propeptide was still present on one of the pro alpha 1(I) chains. In further experiments it was demonstrated that a similar intermediate was seen when a homotrimer of pro alpha 1(I) chains was partially cleaved by the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

The procollagen N-proteinases ADAMTS2, 3 and 14 in pathophysiology

Collagen fibers are the main components of most of the extracellular matrices where they provide a structural support to cells, tissues and organs. Fibril-forming procollagens are synthetized as individual chains that associate to form homo- or hetero-trimers. They are characterized by the presence of a central triple helical domain flanked by amino and carboxy propeptides. Although there are some exceptions, these two propeptides have to be proteolytically removed to allow the almost spontaneous assembly of the trimers into collagen fibrils and fibers. While the carboxy-propeptide is mainly cleaved by proteinases from the tolloid family, the amino-propeptide is usually processed by procollagen N-proteinases: ADAMTS2, 3 and 14.This review summarizes the current knowledge concerning this subfamily of ADAMTS enzymes and discusses their potential involvement in physiopathological processes that are not directly linked to fibrillar procollagen processing.     

Characterization of the amino-terminal segment in type III procollagen.

Native type III collagen and procollagen were prepared from fetal bovine skin. Examination of the cleavage products produced by digestion with tadpole collagenase demonstrated that the three palpha1(III) chains of type III procollagen were linked together by disulfide bonds occurring at both the amino-terminal and carboxy-terminal portions of the molecule. Type III collagen contained interchain disulfide bonds only in the carboxy-terminal region of the molecule. After digestion of procollagen with bacterial collagenase an amino-terminal, triple-stranded peptide fragment was isolated. The reduced and alkylated chain constituents of this fragment had molecular weights of about 21 000. After digestion of procollagen with cyanogen bromide a related triple-stranded fragment was isolated. The chains of the cyanogen bromide fragment had a molecular weight of about 27 000. When the collagenase-derived peptide was fully reduced and alkylated, it became susceptible to further digestion with bacterial collagenase. This treatment released a fragment of about 97 amino acid residues which contained 12 cystein residues and had an amino acid composition typical for globular proteins. A second, non-helical fragment of about 48 amino acid residues contained three cysteines. This latter fragment is formed from sequences that overlap the amino-terminal region in the collagen alpha1(III) chain by 20 amino acids and possesses an antigenic determinant specific for the alpha1(III) chain. The collagenase-sensitive region exposed by reduction comprised about 33 amino acid residues. It was recovered as a mixture of small peptides. These results indicate that the amino-terminal region of type III procollagen has the same type of structure as the homologous region of type I procollagen. It consists of a globular, a collagen-like and a non-helical domain. Interchain disulfide bonding and the occurrence of cysteines in the non-helical domain are, however, unique for type III procollagen.     

Characterization of the procollagen IV cleavage products produced by a specific tumor collagenase

The specific mammalian collagenase isolated from cultures of metastatic mouse PMT sarcoma cells cleaves murine procollagen IV into two segments, of approximate mass ratio 3:1. These fragments were separated by velocity sedimentation, visualized by electron microscopy, and analyzed. The longer COOH-terminal procollagen segment has a 270-nm collagenous portion with a knob at one end. This knob consists of the three previously identified, noncollagenous carboxyl propeptides, of approximately 30,000 daltons each. These carboxyl propeptides are chain-specific, and the three chains of each segment have the same amino to carboxyl orientation. The collagenase cuts through all three chains at one site, and the three-component chains of both the longer COOH-terminal procollagen segment and the shorter NH2-terminal procollagen segment are linked by interchain disulfide bridges. The enzyme cuts off the same COOH-terminal procollagen segment from procollagen IV monomers and tetramers, and the flexibility of this segment is similar to that of interstitial collagen helices. The amino ends of the NH2-terminal procollagen segments derived from tetramers remain joined as the 32-nm long "7 S collagen" junctional complex, and the remaining 89-nm long projecting threads are significantly more flexible than the COOH-terminal procollagen segment. The electrophoretic mobilities of the enzyme cleavage products are consistent with a heterotrimeric composition of this procollagen IV.     

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).     

Role of the pro-α2(I) COOH-terminal region in assembly of type I collagen: Disruption of two intramolecular disulfide bonds in pro-α2(I) blocks assembly of type I collagen

Collagen biosynthesis is a complex process that begins with the association of three procollagen chains. A series of conserved intra- and interchain disulfide bonds in the carboxyl-terminal region of the procollagen chains, or C-propeptide, has been hypothesized to play an important role in the nucleation and alignment of the chains. We tested this hypothesis by analyzing the ability of normal and cysteine-mutated pro-α2(I) chains to assemble into type I collagen heterotrimers when expressed in a cell line (D2) that produces only endogenous pro-α1(I). Pro-α2(I) chains containing single or double cysteine mutations that disrupted individual intra- or interchain disulfide bonds were able to form pepsin resistant type I collagen with pro-α1(I), indicating that individual disulfide bonds were not critical for assembly of the pro-α2(I) chain with pro-α1(I). Pro-α2(I) chains containing a triple cysteine mutation that disrupted both intrachain disulfide bonds were not able to form pepsin resistant type I collagen with pro-α1(I). Therefore, disruption of both pro-α2(I) intrachain disulfide bonds prevented the production and secretion of type I collagen heterotrimers. Although none of the individual disulfide bonds is essential for assembly of the procollagen chains, the presence of at least one intrachain disulfide bond may be necessary as a structural requirement for chain association or to stabilize the protein to prevent intracellular degradation. J.Cell. Biochem. 71:233–242, 1998. © 1998 Wiley-Liss, Inc.     

Assembly of stable human type I and III collagen molecules from hydroxylated recombinant chains in the yeast Pichia pastoris. Effect of an engineered C-terminal oligomerization domain foldon

The C-propeptides of the pro alpha chains of type I and type III procollagens are believed to be essential for correct chain recognition and chain assembly in these molecules. We studied here whether the 30-kDa C-propeptides of the human pC alpha 1(I), pC alpha 2(I), and pC alpha 1(III) chains, i.e. pro alpha chains lacking their N-propeptides, can be replaced by foldon, a 29-amino acid sequence normally located at the C terminus of the polypeptide chains in the bacteriophage T4 fibritin. The alpha foldon chains were expressed in Pichia pastoris cells that also expressed the two types of subunit of human prolyl 4-hydroxylase; the foldon domain was subsequently removed by pepsin treatment, which also digests non-triple helical collagen chains, whereas triple helical collagen molecules are resistant to it. The foldon domain was found to be very effective in chain assembly, as expression of the alpha 1(I)foldon or alpha 1(III)foldon chains gave about 2.5-3-fold the amount of pepsin-resistant type I or type III collagen homotrimers relative to those obtained using the authentic C-propeptides. In contrast, expression of chains with no oligomerization domain led to very low levels of pepsin-resistant molecules. Expression of alpha 2(I)foldon chains gave no pepsin-resistant molecules at all, indicating that in addition to control at the level of the C-propeptide other restrictions at the level of the collagen domain exist that prevent the formation of stable [alpha 2(I)]3 molecules. Co-expression of alpha 1(I)foldon and alpha 2(I)foldon chains led to an efficient assembly of heterotrimeric molecules, their amounts being about 2-fold those obtained with the authentic C-propeptides and the alpha 1(I) to alpha 2(I) ratio being 1.91 +/- 0.31 (S.D.). As the foldon sequence contains no information for chain recognition, our data indicate that chain assembly is influenced not only by the C-terminal oligomerization domain but also by determinants present in the alpha chain domains.