Pro Alpha Chains Research Articles

Enhancement of secretion of human procollagen I in mouse HSP47-expressing insect cells.

We previously demonstrated that insect cells were able to synthesize recombinant human procollagen I as triple-helical heterotrimers when transfected with cDNAs of both proalpha1(I) and proalpha2(I) chains. However, most of the heterotrimers were retained within the cells, unlike in the case of mammalian cells [Tomita, M., Kitajima, T., and Yoshizato, K. (1997) J. Biochem. 1061-1069]. In an attempt to improve the secretion of the heterotrimers, we introduced the putative collagen-specific chaperone HSP47 into this insect expression model. Mouse HSP47 produced by the insect cells bound intracellularly to both human proalpha1(I) and proalpha2(I) chains and enhanced the secretion of procollagen I heterotrimers. HSP47 was also coexpressed with either proalpha1(I) chains or proalpha2(I) chains, which showed that it enhanced the secretion of the former but not the latter. This selective effect of HSP47 was similarly observed in the cells treated with inhibitors of procollagen triple helix formation, indicating that HSP47 can also accelerate the secretion of non-helical procollagens. HSP47 did not change the intracellular solubility of proalpha1(I) and proalpha2(I) chains in 1% NP-40, eliminating the possibility that it prevents proalpha chains from aggregating into insoluble forms within the insect cells. We concluded that HSP47 can play a role in the secretion of alpha1(I)-procollagen chains in the insect cell model. The present study also demonstrated the dissimilarity in the mechanism of folding and secretion of the expressed procollagen I between the insect and mammalian cells.

Angiotensin II stimulates collagen synthesis in human vascular smooth muscle cells. Involvement of the AT(1) receptor, transforming growth factor-beta, and tyrosine phosphorylation.

Angiotensin II is an established regulator of vascular tone and smooth muscle cell (SMC) growth. However, there are little data about its effect on collagen synthesis by SMCs and none regarding the mechanism of such an effect. We studied the effect of angiotensin II on collagen production by human arterial SMCs, using uptake of [(3)H]proline into collagenase-digestible proteins, and by ribonuclease protection assay for mRNA encoding the proalpha1 chain of type I collagen, the major collagen in arteries. This revealed a dose-dependent increase in relative collagen synthesis rate and a dose-dependent increase in proalpha1(I) collagen mRNA abundance, with the half-maximal effect at 1.7 nmol/L. Angiotensin II-stimulated collagen expression was associated with a 6-fold increase in transforming growth factor-beta (TGF-beta) production and was inhibited by a neutralizing antibody to TGF-beta. Both collagen production and TGF-beta release were inhibited by the AT(1)-specific antagonist, losartan, but not by the AT(2) receptor antagonist, PD123319. To determined if tyrosine phosphorylation was functionally linked to collagen synthesis, we studied the effect of 2 mechanistically distinct inhibitors of tyrosine kinase, genistein, and tyrphostin A25. These inhibitors abrogated angiotensin II-mediated procollagen mRNA expression and angiotensin II-mediated TGF-beta production, whereas the inactive homolog tyrphostin A1 had no effect. We conclude that angiotensin II stimulates collagen production in human arterial SMCs via the AT(1) receptor and an autocrine loop of TGF-beta, induction of which requires tyrosine phosphorylation.

Identification of Novel pro-α2(IX) Collagen Gene Mutations in Two Families with Distinctive Oligo-Epiphyseal Forms of Multiple Epiphyseal Dysplasia

Multiple epiphyseal dysplasia (MED) is a genetically heterogeneous disorder with marked clinical and radiographic variability. Traditionally, the mild "Ribbing" and severe "Fairbank" types have been used to define a broad phenotypic spectrum. Mutations in the gene encoding cartilage oligomeric-matrix protein have been shown to result in several types of MED, whereas mutations in the gene encoding the alpha2 chain of type IX collagen (COL9A2) have so far been found only in two families with the Fairbank type of MED. Type IX collagen is a heterotrimer of pro-alpha chains derived from three distinct genes-COL9A1, COL9A2, and COL9A3. In this article, we describe two families with distinctive oligo-epiphyseal forms of MED, which are heterozygous for different mutations in the COL9A2 exon 3/intron 3 splice-donor site. Both of these mutations result in the skipping of exon 3 from COL9A2 mRNA, but the position of the mutation in the splice-donor site determines the stability of the mRNA produced from the mutant COL9A2 allele.

Assembly of the type 1 procollagen molecule: selectivity of the interactions between the alpha 1(I)- and alpha 2(I)-carboxyl propeptides.

Assembly of the heterotrimeric procollagen I molecule is initiated by interactions between the carboxyl propeptide domains of the completed nascent pro alpha chains. The [pro alpha 1(I)]2[pro alpha 2(I)] heterotrimer is the predominant molecule, with much smaller amounts of stable [pro alpha 1(I)]3 homotrimer also being formed. However, the [pro alpha 2(1)]3 homotrimer has not been detected, raising questions as to the mechanism of chain assembly and why [pro alpha2(1)]3 homotrimers are not formed. These questions have been examined here by expressing the intact and amino- or carboxyl-terminal truncated C-propeptides of the pro alpha chains recombinantly in bacteria and in a coupled transcription/translation reticulocyte lysate system. Their interactions were studied in vitro by binding analyses and in vivo by using the yeast two-hybrid system. The C-pro alpha 1(I) interacted with itself, and with C-pro alpha 2(I), as expected. Surprisingly, the C-pro alpha 2(I) also interacted with itself, both in vitro and in vivo. While the interaction of C-pro alpha 2(I) with itself and C-pro alpha 1(I) in vitro was strong, these interactions were weaker in vivo. Deletion of 36 amino acids from the C-terminal domain of C-pro alpha 1 had no effect on its binding to intact self or intact C-pro alpha 2, but the same deletion in C-pro alpha 2 completely abolished its binding to intact C-pro alpha 2 and to C-pro alpha 1. Comparable N-terminal deletions in C-pro alpha 1 or C-pro alpha 2 diminished, but did not abolish, their binding to intact C-pro alpha 1 and C-pro alpha 2. In the yeast two-hybrid system, C-pro alpha 2 interacted with itself more weakly than with C-pro alpha 1. Molecular modeling and circular dichroism analyses showed that C-pro alpha 1 and C-pro alpha 2 have different folded structures and stability. Studies with antibodies specific to the C-pro alpha1 and alpha2 peptides showed them to precipitate different, specific, and distinct cell proteins from fibroblast lysates. The C-pro alpha 2(I) antibody complexed with more cell proteins. We hypothesize that the lack of pro alpha 2(I) homotrimers is not due to the inability of the C-pro alpha 2(I) to interact with itself, but rather to the competing presence of other cell proteins. The specificity of these interactions may reside in conformational differences in N- and C-terminal sequences of the two propeptides or in their different folding patterns.

Expression of an engineered form of recombinant procollagen in mouse milk.

We have examined the suitability of the mouse mammary gland for expression of novel recombinant procollagens that can be used for biomedical applications. We generated transgenic mouse lines containing cDNA constructs encoding recombinant procollagen, along with the alpha and beta subunits of prolyl 4-hydroxylase, an enzyme that modifies the collagen into a form that is stable at body temperature. The lines expressed relatively high levels (50-200 micrograms/ml) of recombinant procollagen in milk. As engineered, the recombinant procollagen was shortened and consisted of a pro alpha 2(I) chain capable of forming a triple-helical homotrimer not normally found in nature. Analysis of the product demonstrated that (1) the pro alpha chains formed disulphide-linked trimers, (2) the trimers contained a thermostable triple-helical domain, (3) the N-propeptides were aligned correctly, and (4) the expressed procollagen was not proteolytically processed to collagen in milk.

A Point Mutation in an Intronic Branch Site Results in Aberrant Splicing of COL5A1 and in Ehlers-Danlos Syndrome Type II in Two British Families

Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective-tissue disorders characterized by skin fragility, joint laxity, and skeletal deformities. Type V collagen appears to have a causal role in EDS types I and II, which show phenotypic overlap and may sometimes be allelic. Type V collagen can exist as a heterotrimer, [alpha1(V)]2alpha2(V), and it both coassembles with and regulates type I collagen-fibril diameter. Using an intragenic COL5A1 polymorphism, we have demonstrated linkage, at zero recombination, to the same allele in two large British EDS type II families (LOD scores 4.1 and 4.3). Affected members from each family were heterozygous for a point mutation in intron 32 (IVS32:T-25G), causing the 45-bp exon 33 to be lost from the mRNA in approximately 60% of transcripts from the mutant gene. This mutation lies only 2 bp upstream of a highly conserved adenosine in the consensus branch-site sequence, which is required for lariat formation. Although both families shared the same marker allele, we have been unable to identify a common genealogy. This is the first description of a mutation at the lariat branch site, which plays a pivotal role in the splicing mechanism, in a collagen gene. Very probably, the resulting in-frame exon skip has a dominant-negative effect due to incorporation of the mutant proalpha chain into the triple-helical molecule. These findings further confirm the importance of type V collagen in the causation of EDS type II, and the novel collagen mutation indicates the importance of the lariat branch site in splicing.

Expression and characterization of recombinant human type II collagens with low and high contents of hydroxylysine and its glycosylated forms.

Insect cells coinfected with two baculoviruses, one coding for the pro alpha chains of human type II procollagen and the other for both the alpha and beta subunits of human prolyl 4-hydroxylase, produced the cartilage-specific type II collagen with a stable triple helix. The highest expression levels, up to 50 mg/l of type II collagen, were obtained in suspension culture using a modified construct in which sequences coding for the signal peptide and N propeptide of type II procollagen had been replaced by those for type III procollagen. The type III N propeptide artificially generated into type II procollagen was found to be cleaved at a much higher rate than the wild-type type II N propeptide, probably because the former interacted poorly with the triple-helical domain of type II procollagen. The amino acid composition of the recombinant type II collagen was very similar to that of the non-recombinant protein, but the hydroxylysine content was only 17% and that of glycosylated hydroxylysines was equally low. The hydroxylysine content was increased to the level found in the non-recombinant collagen by using an additional baculovirus coding for lysyl hydroxylase, and a substantial increase was also found in the glycosylated hydroxylysine content. No difference in thermal stability was found between the low- and high-hydroxylysine collagens.

Expression of Wild-Type and Modified Proα Chains of Human Type I Procollagen in Insect Cells Leads to the Formation of Stable [α1(I)]2α2(I) Collagen Heterotrimers and [α1(I)]3 Homotrimers but Not [α2(I)]3 Homotrimers

Insect cells coinfected with a baculovirus coding for the proalpha1(I) chain of human type I procollagen and a double promoter virus coding for the alpha and beta subunits of human prolyl 4-hydroxylase produced homotrimeric [proalpha1(I)]3 procollagen molecules. The use of an additional virus coding for the proalpha2(I) chain led to the formation of a heterotrimeric molecule with the correct 2:1 ratio of proalpha1 to proalpha2 chains of type I procollagen (proalpha1(I) and proalpha2(I) chains, respectively), unless the proalpha1(I) chain was expressed in a relatively large excess. Replacement of the sequences coding for the signal peptide and the N propeptide of the proalpha1(I) chain with those of the proalpha1(III) chain increased level of expression of the proalpha1(I) chain, whereas no similar effect was found when the corresponding modification was made to the virus coding for the proalpha2(I) chain. Molecules containing such modified N propeptides were found to be processed at their N terminus more rapidly than those containing the wild-type propeptides. The Tm of the type I collagen homotrimer was similar to that of the heterotrimer, both values being about 42-43 degrees C when determined by circular dichroism. The wild-type proalpha2(I) chain formed no homotrimers. Replacement of the C propeptide of the proalpha2(I) chain with that of the proalpha1(I) chain or proalpha1 chain of type III procollagen (proalpha1(III) chain) led to the formation of homotrimers, but the alpha2(I) chains in such molecules were completely digested by pepsin in 1 h at 22 degrees C. The data thus suggest that, in addition to control at the level of the C propeptide, other restrictions may exist at the level of the collagen domain that prevent the formation of stable homotrimeric [proalpha2(I)]3 molecules in insect cells.

Formation of recombinant human procollagen I heterotrimers in a baculovirus expression system.

The present study describes the production of human procollagen I in a baculovirus expression system. Recombinant baculovirus carrying pro alpha1(I) or pro alpha2(I) cDNA was constructed and infected to Sf9 cells. Full-length pro alpha1(I) or pro alpha2(I) chains were synthesized by the cells infected with either of the recombinant viruses. The pro alpha1(I) chains formed pepsin-resistant homotrimers stabilized by interchain disulfide bonds, a small proportion of which was secreted into the culture medium. The pro alpha2(I) chains were not linked into trimers by disulfide bonds and failed to form stable triple helices, although some chains were suggested to exist as dimers or unstable trimers in which only two chains were linked by disulfide bonds. In spite of their non-helicity, the pro alpha2(I) chains were secreted at a higher rate than the pro alpha1(I) chains. Sf9 cells simultaneously synthesized both pro alpha1(I) and pro alpha2(I) chains when the cells were co-infected with the two recombinant viruses. Pepsin-treatment of the product clearly demonstrated the production of procollagen I heterotrimers composed of two pro alpha1(I) chains and one pro alpha2(I) chain, homotrimers of the pro alpha1(I) chains being negligible. This expression system appears to offer a unique means of studying the mechanism of chain association and secretion during procollagen biosynthesis.

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.

Extensive Alternative Splicing within the Amino-propeptide Coding Domain of α2(XI) Procollagen mRNAs: EXPRESSION OF TRANSCRIPTS ENCODING TRUNCATED PRO-α CHAINS

Heterogeneity in type XI procollagen structure is extensive because all three alpha(XI) collagen genes undergo complex alternative splicing within the amino-propeptide coding domain. Exon 7 of the human and exons 6-8 of the mouse alpha2(XI) collagen genes, encoding part of the amino-propeptide variable region, have recently been shown to be alternatively spliced. We show that exon 6-containing mRNAs for human alpha2(XI) procollagen are expressed at 28 weeks in fetal tendon and cartilage but not at 38-44 days or 11 weeks. In the mouse, exon 6 is expressed in chondrocytes from 13.5 days onward. We recently identified conserved sequences within intron 6 of the human and mouse alpha2(XI) collagen genes, containing additional consensus splice acceptor and donor sites that potentially increase the size of exon 7, dividing it into three parts, designated 7A, 7B, and 7C. We show by reverse transcription polymerase chain reaction and in situ hybridization that these potential splice sites are used to yield additional alpha2(XI) procollagen mRNA splice variants that are expressed in fetal tissues. In human, expression of exon 7B-containing transcripts may be developmental stage-specific. Interestingly, inclusion of exon 7A or exon 7B in human and mouse alpha2(XI) procollagen mRNAs, respectively, would result in the insertion of an in-frame termination codon, suggesting that some of the additional splice variants encode a truncated pro-alpha2(XI) chain.

Characterization of Human Type III Collagen Expressed in a Baculovirus System: PRODUCTION OF A PROTEIN WITH A STABLE TRIPLE HELIX REQUIRES COEXPRESSION WITH THE TWO TYPES OF RECOMBINANT PROLYL 4-HYDROXYLASE SUBUNIT

An efficient expression system for recombinant collagens would have numerous scientific and practical applications. Nevertheless, most recombinant systems are not suitable for this purpose, as they do not have sufficient amounts of prolyl 4-hydroxylase activity. Pro-alpha 1 chains of human type III collagen expressed in insect cells by a baculovirus vector are reported here to contain significant amounts of 4-hydroxyproline and to form triple-helical molecules, although the Tm of the triple helices was only about 32-34 degrees C. Coexpression of the pro-alpha1(III) chains with the alpha and beta subunits of human prolyl 4-hydroxylase increased the Tm to about 40 degrees C, provided that ascorbate was added to the culture medium. The level of expression of type III procollagen was also increased in the presence of the recombinant prolyl 4-hydroxylase, and the pro-alpha 1(III) chains and alpha1(III) chains were found to be present in disulfide-bonded molecules. Most of the triple-helical collagen produced was retained within the insect cells and could be extracted from the cell pellet. The highest expression levels were obtained in High Five cells, which produced up to about 80 microg of cellular type III collagen (120 microg of procollagen) per 5 X 10(6) cells in monolayer culture and up to 40 mg/liter of cellular type III collagen (60 mg/liter procollagen) in suspension. The 4-hydroxyproline content and Tm of the purified recombinant type III collagen were very similar to those of the nonrecombinant protein, but the hydroxylysine content was slightly lower, being about 3 residues/1000 in the former and 5/1000 in the latter.

Posttranscriptional aspects of the biosynthesis of type 1 collagen pro-alpha chains: the effects of posttranslational modifications on synthesis pauses during elongation of the pro alpha 1 (I) chain.

Early studies indicated that chain elongation pauses were prominent during the in vivo synthesis of type I procollagen chains, and it was postulated [Kirk et al., (1987): J Biol Chem 262:5540-5545.] that these might have a role in the coordination of procollagen I molecular assembly. To examine this postulate, polysomes isolated from [(14)C]-Pro-labeled 3T6 cells were subjected to SDS-PAGE. The resulting gels were Western blotted and screened with a monoclonal antibody (SP1 .D8) directed against the N-terminal region of the pro alpha 1 (I) chain. The blots were fluorographed, which also permitted analysis of the pro alpha 2 (I) chain. There was a prominent pro alpha1 synthesis pause near the completion of full-length chain elongation, not matched by a pro alpha 2 pause. The amount of labeled polysome-associated near-full length pro alpha 1 (I) chains increased in parallel with labeling time. After 24 h in culture -[(14)C-Pro], collagen synthesis ceased but unlabeled polysome-associated pro alpha1 chains were readily detected by SP1 .D8. Change to fresh culture medium +[(14)C-Pro] reinitiated synthesis and permitted tracing of the newly synthesized labeled pro a chains through the polysome and intracellular compartments. The secreted procollagen molecules had a 2:1 pro alpha 1 (1):pro alpha 2 (I) chain ratio but the polysome-bound peptides did not. Pulse-chase experiments showed that near-full length pro alpha 1 (I) chains remained bound to polysomes as long as 4 h after reinitiation of translation but there was no evidence for pro alpha 2 (I) chain accumulation. The hydroxylation inhibitor alpha, alpha'-dipyridyl, and triple-helix inhibitors cis-hydroxyproline and 3,4 dehydroproline had minimal effects on the buildup of polysome-associated pro al chains. The glycosylation inhibitor tunicamycin also failed to change the final pro alpha 1 chain pausing, but it did cause the appearance of several discrete lower molecular weight pro alpha 1-related polypeptides that could not be accounted for simply as the result of lack of N-linked glycosylation in the C-propeptide regions. Disulfide bond experiments showed that some of the paused nascent polysome-associated pro alpha 1 (I) chains were disulfide bonded. Thus, while synthesis of pro alpha 1 (I) and pro alpha 2 (I) chains proceeds in parallel within the same ER compartments, their elongation rates are not coordinated. Interactions leading to heterotrimer formation are a late event which may affect the rate of release of the completed pro alpha 1 (I) chain from the polysome. The release of completed nascent pro alpha 1 (I) chains from their polysomal complexes is regulated by a mechanism not operating in the synthesis of pro alpha 2 (I) chains. The pro alpha 1 (I) chain release process is not connected directly with hydroxylation, glycosylation or triple-helix formation.

Endoplasmic reticulum protein Hsp47 binds specifically to the N-terminal globular domain of the amino-propeptide of the procollagen I alpha 1 (I)-chain.

Hsp47, an endoplasmic reticulum-resident heat shock protein in fibroblasts, has gelatin-binding properties. It had been hypothesized that it functions as a chaperone regulating procollagen chain folding and/or assembly, but the mechanism of the hsp47-procollagen I interaction was not clear. Hsp47 could bind to both denatured and native procollagen I. A series of competition studies were carried out in which various collagens and collagen domain peptides were incubated with 35[S]-methionine-labeled murine 3T6 cell lysates prior to mixing with gelatin-Sepharose 4B beads. The gelatin-bound proteins were collected and analyzed by gel electrophoresis and autoradiography. Collagenase digested procollagen I had the same effect as denatured intact procollagen, indicating that the propeptides were the major interaction sites. The addition of intact pro alpha 1(I)-N-propeptide at 25 micrograms/ml completely inhibited hsp47 binding to the gelatin-Sepharose. Even the pentapeptide VPTDE, residues 86-90 of the pro alpha 1(I)-N-propeptide, inhibits hsp47-gelatin binding. These data implicating the pro alpha 1(I)-N-propeptide domain were confirmed by examination of polysome-associated pro alpha chains. The nascent pro alpha 1(I)-chains with intact N-propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti-pro alpha 1 (I)-N-propeptide antibody SP1.D8 unless dissociated from the hsp47. GST-fusion protein constructs of residues 23-108 (NP1), 23-151 (NP2), and 23-178 (NP3) within the pro alpha 1 (I)- N-propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysates. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane-free in vitro cotranslation systems in which the complete pro alpha 1(I)- and pro alpha 2(I)-chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro alpha 2(I)-chains and hsp47, whereas there was strong interaction between pro alpha 1(I)-chains and nascent hsp47. SP1.D8 could not precipitate pro alpha 1(I)-chains from the translation mix if nascent hsp47 was present. These data all suggest that if hsp47 has a "chaperone" role during procollagen chain processing and folding it performs this specific role via its preferential interaction with the pro alpha 1 (I) chain, and the pro alpha 1(I) amino-propeptide region in particular.

Relationships between translation of pro α1(I) and pro α2(I) mRNAs during synthesis of the type I procollagen heterotrimer

Final assembly of the procollagen I heterotrimeric molecule is initiated by interactions between the carboxyl propeptide domains of completed, or nearly completed nascent pro alpha chains. These interactions register the chains for triple helix folding. Prior to these events, however, the appropriate nascent chains must be brought within the same compartments of the endoplasmic reticulum (ER). We hypothesize that the co-localization of the synthesis of the nascent pro alpha1(I) and pro alpha2(I) chains results from an interaction between their translational complexes during chain synthesis. This has been investigated by studying the polyribosomal loading of the pro alpha-chain messages during in vitro translation in the presence and absence of microsomal membranes, and in cells which have the ability to synthesize the pro alpha1 homotrimer or the normal heterotrimer. Recombinant human pro alpha1(I) and pro alpha2(I) cDNAs were inserted into plasmids and then transcribed in vitro. The resulting RNAs were translated separately and in mixture in a cell-free rabbit reticulocyte lysate +/- canine pancreatic microsomes. Cycloheximide (100 mu g/ml) was added and the polysomes were collected and fractionated on a 15-50% sucrose gradient. The RNA was extracted from each fraction and the level of each chain message was determined by RT-PCR. Polysomes from K16 (heterotrimer-producing), W8 (pro alpha1(I) homotrimer), and A2' (heterotrimer + homotrimer) cells were similarly analyzed. Translations of the pro alpha1(I) and pro alpha2(I) messages proceeded independently in the cell-free, membrane-free systems, but were coordinately altered in the presence of membrane. The cell-free + membrane translation systems mimicked the behavior of the comparable cell polysome mRNA loading distributions. These data all suggest that there is an interaction between the pro alpha chain translational complexes at the ER membrane surface which temporally and spatially localize the nascent chains for efficient heteromeric selection and folding.

The Human COL11A2 Gene Structure Indicates that the Gene Has Not Evolved with the Genes for the Major Fibrillar Collagens

The human COL11A2 gene was analyzed from two overlapping cosmid clones that were previously isolated in the course of searching the human major histocompatibility region (Janatipour, M., Naumov, Y., Ando, A., Sugimura, K., Okamoto, N., Tsuji, K., Abe, K., and Inoko, H. (1992) Immunogenetics 35, 272-278). Nucleotide sequencing defined over 28,000 base pairs of the gene. It was shown to contain 66 exons. As with most genes for fibrillar collagens, the first intron was among the largest, and the introns at the 5'-end of the gene were in general larger than the introns at the 3'-end. Analysis of the exons coding for the major triple helical domain indicated that the gene structure had not evolved with the genes for the major fibrillar collagens in that there were marked differences in the number of exons, the exon sizes, and codon usage. The gene was located close to the gene for the retinoic X receptor beta in a head-to-tail arrangement similar to that previously seen with the two mouse genes (P. Vandenberg and D. J. Prockop, submitted for publication). Also, there was marked interspecies homology in the intergenic sequences. The amino acid sequences and the pattern of charged amino acids in the major triple helix of the alpha 2(XI) chain suggested that the chain can be incorporated into the same molecule as alpha 1(XI) and alpha 1(V) chains but not into the same molecule as the alpha 3(XI)/alpha 1(II) chain. The structure of the carboxyl-terminal propeptide was similar to the carboxyl-terminal propeptides of the pro alpha 1(XI) chain and pro alpha chains of other fibrillar collagens, but it was shorter because of internal deletions of about 30 amino acids.

Perinatal lethal osteogenesis imperfecta.

Perinatal lethal osteogenesis imperfecta is the result of heterozygous mutations of the COL1A1 and COL1A2 genes that encode the alpha 1(I) and alpha 2(I) chains of type I collagen, respectively. Point mutations resulting in the substitution of Gly residues in Gly-X-Y amino acid triplets of the triple helical domain of the alpha 1(I) or alpha 2(I) chains are the most frequent mutations. They interrupt the repetitive Gly-X-Y structure that is mandatory for the formation of a stable triple helix. Most babies have their own private de novo mutation. However, the recurrence rate is about 7% owing to germline mosaicism in one parent. The mutations act in a dominant negative manner as the mutant pro alpha chains are incorporated into type I procollagen molecules that also contain normal pro alpha chains. The abnormal molecules are poorly secreted, more susceptible to degradation, and impair the formation of the extracellular matrix. The collagen fibres are abnormally organised and mineralisation is impaired. The severity of the clinical phenotype appears to be related to the type of mutation, its location in the alpha chain, the surrounding amino acid sequences, and the level of expression of the mutant allele.

Mutations in type 1 procollagen that cause osteogenesis imperfecta: effects of the mutations on the assembly of collagen into fibrils, the basis of phenotypic variations, and potential antisense therapies.

Work by a large number of investigators over the last decade has established that over 90% of patients with osteogenesis imperfecta have mutations in one of the two genes for type I procollagen, that most unrelated probands have different mutations in the genes, and that the mutations found in most of the serious variants of the disease cause synthesis of abnormal pro alpha chains of the protein. The results have demonstrated that synthesis of structurally abnormal but partially functional pro alpha chains can interfere with folding of the central region of the protein into a triple-helical conformation, prevent processing of the N-terminal propeptides of procollagen, or produce subtle alterations in conformation that interfere with the self-assembly of the protein into collagen fibrils. One of the unsolved mysteries about the disease is why some mutations produce severe phenotypes, whereas very similar mutations produce mild phenotypes. Recent studies in transgenic mice suggest that nongenetic factors, such as stochastic events during development, may determine the severity of the disease phenotype produced by a specific mutation. Also, recent results raised the possibility that strategies of antisense gene therapy may be effective in treating the disease some time in the future. Specific inhibition of expression of a mutated collagen gene has been obtained with antisense oligonucleotides in cell culture experiments. However, there is no means of selective delivery of antisense oligonucleotides to the appropriate tissues.

BiP binds type I procollagen pro alpha chains with mutations in the carboxyl-terminal propeptide synthesized by cells from patients with osteogenesis imperfecta.

Of 20 fibroblast cell strains from patients with osteogenesis imperfecta (OI), a disease caused by mutations in the genes encoding type I procollagen, three had increased synthesis of BiP (GRP78), an hsp70-related, endoplasmic reticulum-resident protein. All three strains carry unique mutations in pro alpha 1(I) chains which impair type I procollagen chain association. Immunoprecipitation and pulse-chase experiments show that BiP (immunoglobulin heavy chain-binding protein) stably binds pro alpha 1(I) chains in these three cell strains after a brief lag. Ascorbate, which increases procollagen synthesis, increases BiP synthesis and content in these three strains and not in the others. In one of these three strains, BiP content is constitutively elevated prior to ascorbate treatment, and BiP is less inducible. This strain also has relatively high levels of synthesis and content of GRP94, another endoplasmic reticulum-resident stress protein. Pretreating each of the three cell strains to increase their BiP content reduces subsequent ascorbate-mediated BiP induction. BiP synthesis in the 17 other OI strains examined, which had a variety of type I procollagen mutations, was normal. These results suggest that BiP is induced by and binds procollagen with specific types of mutations: ones in the carboxyl-terminal propeptide that interfere with chain association. The recognition by BiP of such procollagen in OI cell strains shows that BiP plays a role in the physiological response to the production of some disease-producing abnormal proteins.

Rat glioma cell lines C6 and 9L synthesize type 1 collagen in vitro

Glial cell lines (C6, a glioma and 9L, a gliosarcoma) grown in vitro produce type 1 collagen which is detectable in the extracellular matrix by immunocytochemistry. Northern blot analysis using a cDNA specific for the proalpha2 (I) chain of procollagen indicates the presence of a single transcript with an apparent size of 4.8 kb in the C6 cell line, whereas two transcripts with apparent sizes of 5.8 and 4.8 kb are visualized in the 9L cells. The stimulatory effect of ascorbic acid on collagen production is detectable by a 20–27% increase in the concentration of hydroxyproline in the culture medium from the two glioma cell lines. Therefore these glioma cell lines provide a valuable model system for comparative investigations on the regulation of type 1 collagen synthesis by nonmesenchymal cells of neuroepithelial origin.