Biomedical subjects
D J Prockop
Publications and source records attributed to D J Prockop.
The gene for the alpha 1(IV) chain of human type IV procollagen: the exon structures do not coincide with the two structural subdomains in the globular carboxy-terminus of the protein.
Previous studies on the coding sequences of DNAs for the alpha 1(IV) chain of basement membrane collagen demonstrated a striking homology between the first 115 and the second 114 amino acids of the globular (NC1) domain of the protein. Also, alignment of the 12 cysteine residues indicated that the homology was particularly strong around three paired clusters of amino acids around cysteine residues. Here we have isolated a cosmid clone containing the 3'-end of the gene. Analysis of the clone and previously isolated lambda clones demonstrated that the intron--exon patterns of the gene does not reflect the homology in the protein. Therefore the homology cannot have arisen in any simple manner from gene duplications.
Assessment of small polymorphisms in defined human collagen gene segments.
Restriction fragment length polymorphisms (RFLPs) greater than 1.5 kb in size have been identified in all but one of the human fibrillar collagen genes. However, the number of informative RFLPs for these genes is still limited. Here we present the conjunct use of two techniques for the assessment of small length variations within defined segments of the genome. This strategy has the potential to be used for the allelic exclusion of cloned genomic fragments prior to sequencing. Moreover it can be a useful and sensitive tool to determine if genetic linkage exists between abnormal phenotypes and loci where conventional Southern blotting analysis has failed to detect informative RFLPs.
Large introns in the 3' end of the gene for the pro alpha 1 (IV) chain of human basement membrane collagen.
Using a recently characterized cDNA clone (HT-21) coding for the pro alpha 1 (IV) chain of human type IV procollagen, we have isolated three clones from a bacterio-phage lambda Charon 4A library of human genomic DNA. The intron/exon structure of the pro alpha 1 (IV) genomic clones was analyzed by heteroduplex electron microscopy and nucleotide sequencing. The analysis showed that the introns separating exons 2-9 are large and have a total length of over 12,000 base pairs (bp). Six of seven exons at the 3' end of the gene coded for -Gly-Xaa-Yaa-repeats of the collagenous part of the chain. Five of the -Gly-Xaa-Yaa- coding exons (numbers 5-9) varied in size between 72 bp and 134 bp, and none of them were 54 bp or multiples thereof. A sixth exon (exon 4) was a junction exon containing 71 bp coding for -Gly-Xaa-Yaa- sequences and 142 bp coding for the carboxyl-terminal noncollagenous domain (NC-1). The seventh exon (exon 3, 178 bp) coded for sequences of the NC-1 domain. Five of the six -Gly-Xaa-Yaa- coding exons began with the second base coding for glycine, and only one exon began with a complete glycine codon at the 5' end. The results (i) suggest that the gene for the pro alpha 1(IV) chain of human basement membrane collagen is significantly larger than the genes for fibrillar collagens and (ii) show that it lacks the 54-bp exon repeats characteristic of fibrillar collagen genes.
Connective tissue biochemistry of neurofibromas.
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Type I procollagen carboxyl-terminal proteinase from chick embryo tendons. Purification and characterization.
Procollagen carboxyl-terminal proteinase, the enzyme which cleaves the carboxyl-terminal propeptides from type I procollagen, was extensively purified in a yield of 25% from pooled culture media of 17-day-old chick embryo tendons using a procedure which involved chromatography on Green A Dye matrix gel, concanavalin A-Sepharose and heparin-Sepharose, and filtration gels of Sephacryl S-300 and S-200. The purified enzyme is a neutral, Ca2+-dependent proteinase which is inhibited by metal chelators, but not by inhibitors for serine and cysteine proteinases. Calcium in a concentration of 5-10 mM is required for optimal activity. The molecular weight of the enzyme was determined to be 97,000-110,000 by gel filtration and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Other properties of the carboxyl-terminal proteinase are: 1) the Km for the type I procollagen is 96 nM at pH 7.5 and 35 degrees C; 2) the activation energy for the reaction with type I procollagen is 21,000 cal mol-1; 3) amino acid sequencing of the released carboxyl-terminal propeptide indicated the enzyme specifically cleaves an -Ala-Asp- bond in both the pro-alpha 1(I) and pro-alpha 2(I) chains; 4) the enzyme specifically cleaves the carboxyl-terminal propeptides of a homotrimer of pro-alpha 1(I) chains and type II and III procollagens, but it does not cleave type IV procollagen. The results suggest that the enzyme is involved in the processing of type I procollagen in vivo.
cDNA clones coding for the pro-alpha1(IV) chain of human type IV procollagen reveal an unusual homology of amino acid sequences in two halves of the carboxyl-terminal domain.
We report the isolation and characterization of cDNA clones coding for part of the pro-alpha1(IV) chain of human type IV procollagen. A cDNA library was prepared from total RNA isolated from a cultured human tumor cell line, HT-1080, and screened with a cloned mouse cDNA coding for the pro-alpha1(IV) chain. The largest cDNA clone encoded for 185 amino acid residues of the -Gly-X-Y-sequence of the human pro-alpha1(IV) chain, all of the globular carboxyl-terminal domain, and the 3' noncoding region. The results provide the first complete sequence for the carboxyl-terminal globular portion of a type IV procollagen chain. A striking feature of the carboxyl-terminal globular domain was a homology between the first and second half of the structure. The homology involved all 12 cysteine residues, the spacing between the cysteine residues, and many adjacent amino acids. The results raised the possibility that evolution of the globular domain involved duplication of an ancestral sequence coding for about 100 amino acids, 6 of which were cysteine. The homology, however, was more apparent in the amino acid sequence than in the nucleotide sequence, and, therefore, the results suggested that the homology reflects selective pressure on the function of the protein more than conservation of the nucleotide sequences in the gene. The nucleotide sequences of the 3' noncoding region of the cDNAs contained four polyadenylation signals of AATAAA. Three or four of the polyadenylation signals were probably used in transcription, since one major and two minor smaller RNA species from human skin fibroblasts hydridized with the cDNAs. In further studies, sorted human chromosomes were used to locate the gene for the pro-alpha1(IV) chain on chromosome 13.
Synthesis of an altered type III procollagen in a patient with type IV Ehlers-Danlos syndrome. A structural change in the alpha 1(III) chain which makes the protein more susceptible to proteinases.
The synthesis of type III procollagen was examined in cultured fibroblasts from ten patients with type IV Ehlers-Danlos syndrome, a heritable disorder of connective tissue. With fibroblasts from nine patients, a decreased amount of labeled type III procollagen was recovered in the medium after the cells were incubated with radioactive amino acids for 24 h. The results were compatible with undefined defects in type III procollagen. The culture medium from one patient contained apparently normal amounts of type III procollagen after a 24-h labeling. However, the pro-alpha 1(III) chains from the medium of the patient's fibroblasts appeared as an abnormally broad band when examined by gel electrophoresis in sodium dodecyl sulfate. Analysis of fragments generated by vertebrate collagenase and cyanogen bromide located a structural defect between amino acid residues 555 and 775 in half of the alpha 1(III) chains. Most of the patient's type III procollagen was susceptible to digestion by pepsin or a mixture of chymotrypsin and trypsin at temperatures at which normal type III procollagen resisted digestion. Cyanogen bromide digestion of samples of the patient's skin revealed that the amount of type III was reduced more than 4-fold. The results support the hypothesis that both normal and structurally altered pro-alpha 1(III) chains are being incorporated into type III procollagen synthesized by the patient's fibroblasts and that type III procollagen molecules containing one, two, or three structurally altered pro-alpha 1(III) chains are rapidly degraded by proteinases in the tissues.
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)
Type I procollagen N-proteinase from whole chick embryos. Cleavage of a homotrimer of pro-alpha 1(I) chains and the requirement for procollagen with a triple-helical conformation.
Procollagen N-proteinase, the enzyme which cleaves the NH2-terminal propeptides from type I procollagen, was purified over 15,000-fold from extracts of chick embryos by chromatography on columns of DEAE-cellulose, concanavalin A-agarose, heparin-agarose, pN-collagen-agarose, and a filtration gel. The purified enzyme had an apparent molecular weight of 320,000 as estimated by gel filtration and a pH optimum for activity of 7.4 to 9.0. The enzyme was inhibited by metal chelators and the thiol reagent dithiothreitol. Addition of calcium was required for maximal activity under the standard assay conditions, and the presence of calcium decreased thermal inactivation at 37 degrees C. The purified enzyme cleaved a homotrimer of pro-alpha 1(I) chains, an observation which indicated that the presence of pro-alpha 2(I) chain is not essential for the enzymic cleavage of NH2-terminal propeptides. Previous observations suggesting that the enzyme requires a substrate with a native conformation were explored further by reacting the enzyme with type I procollagen at different temperatures. Type I procollagen from chick embryo fibroblasts became resistant to cleavage at about 43 degrees C. Type I procollagen from human skin fibroblasts, which was previously shown to have a slightly lower thermal stability than chick embryo type I procollagen, became resistant to cleavage at temperatures that were about 2 degrees C lower. The results suggested that the enzyme is a sensitive probe for the three-dimensional structure of the NH2-terminal region of the procollagen molecule and that it requires the protein substrate to be triple helical.
Altered helical structure of a homotrimer of alpha 1(I)chains synthesized by fibroblasts from a variant of osteogenesis imperfecta.
Cultured skin fibroblasts from a variant of osteogenesis imperfecta were previously shown to synthesize a type I procollagen which was a homotrimer of pro alpha 1(I) chains. Trimers of alpha 1(I) collagen were isolated by pepsin digestion of culture medium from these fibroblasts. The amino acid composition of the isolated protein indicated that it contained an increased amount of hydroxylysine, apparently because of post-translational over-modification. The thermal stability of the alpha 1(I) trimers was examined by circular dichroism. We found no consistent difference in the melting curve of the alpha 1(I) trimers compared to control type I collagen. We next examined the thermal stability of the alpha 1(I) trimers using digestion with a combination of trypsin and alpha-chymotrypsin as an alternative probe of helical stability. When enzymatic digestions were carried out at 36 degrees to 40 degrees C, the alpha 1(I) chains in the trimers were cleaved to polypeptides which were shortened by approximately 100 amino acids. Vertebrate collagenase digestion of the shortened molecules indicated that the 100 amino acid segment removed from each alpha 1(I) chain was located at the carboxyl-terminus. The decreased thermal stability of the alpha 1(I) trimers was probably explained by the absence of alpha 2(I) chains in the molecules. The results, however, did not exclude the possibility that the post-translational over-modification of the alpha 1(I) chains contributed to the altered helical structure.
Mutations in osteogenesis imperfecta leading to the synthesis of abnormal type I procollagens.
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Mutations in collagen genes. Consequences for rare and common diseases.
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Modulation of procollagen gene expression by retinoids. Inhibition of collagen production by retinoic acid accompanied by reduced type I procollagen messenger ribonucleic acid levels in human skin fibroblast cultures.
Recent clinical observations have suggested that retinoids, which are in frequent use in dermatology, can affect the connective tissue metabolism in skin and other tissues. In this study, we examined the effects of several retinoids on the metabolism of collagen by human skin fibroblasts in culture. Incubation of cultured fibroblasts with all-trans-retinoic acid or 13-cis-retinoic acid, in 10(-5) M or higher concentrations, markedly reduced the procollagen production, as measured by synthesis of radioactive hydroxyproline. The effect was selective in that little, if any, inhibition was noted in the incorporation of [3H]leucine into the noncollagenous proteins, when the cells were incubated with the retinoids in 10(-5) M concentration. Similar reduction in procollagen production was noted with retinol and retinal, whereas an aromatic analogue of retinoic acid ethyl ester (RO-10-9359) resulted in a slight increase in procollagen production in these cultures. The reduction in procollagen production by all-trans-retinoic acid was accompanied by a similar reduction in pro alpha 2(I) of type I procollagen specific messenger RNA (mRNA), as detected by dot blot and Northern blot hybridizations. Hybridizations with human fibronectin and beta-actin specific DNA probes indicated that the levels of the corresponding mRNAs were not affected by the retinoids, further suggesting selectivity in the inhibition of procollagen gene expression. Further control experiments indicated that all-trans-retinoic acid, under the culture conditions employed, did not affect the posttranslational hydroxylation of prolyl residues, the mannosylation of newly synthesized procollagen, the specific radioactivity of the intracellular prolyltransfer RNA pool, or DNA replication. All-trans-retinoic acid also elicited a reduction in trypsin-activatable collagenase, but not in the activity of prolyl hydroxylase or an elastaselike neutral protease in the fibroblast cultures. Incubation of three fibroblast lines established from human keloids with all-trans-retinoic acid or 13-cis-retinoic acid also resulted in a marked reduction in procollagen production. The results, therefore, suggest that further development of retinoids might provide a novel means of modulating collagen gene expression in patients with various diseases affecting the connective tissues.
Collagen synthesis and collagenase activity in dermal fibroblasts from patients with diabetes and digital sclerosis.
Collagen production and collagenase activity were measured in dermal fibroblast cultures obtained from eight patients with diabetes with digital sclerosis and three normal controls. Total collagen synthesis in patients with diabetes was reduced in comparison with controls. DNA replication was also reduced in patients with diabetes. No differences in collagenase activity were noted. Our results suggest that, in contrast to systemic sclerosis, increased synthesis does not contribute to the collagen accumulation of diabetic digital sclerosis. Decreased degradation related to nonenzymatic glucosylation of collagen is a more likely mechanism.
DNA polymorphisms in collagen genes: potential use in the study of disease.
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A heterozygous defect for structurally altered pro-alpha 2 chain of type I procollagen in a mild variant of osteogenesis imperfecta. The altered structure decreases the thermal stability of procollagen and makes it resistant to procollagen N-proteinase.
Cultured skin fibroblasts from a proband with an autosomal dominant variant of osteogenesis inperfecta were found to synthesize approximately equal amounts of normal pro-alpha 2(I) chains of type I procollagen and pro-alpha 2(I) chains which migrated more rapidly when examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The structural alteration was present in alpha 2(I)-CB4, a cyanogen bromide fragment containing amino acid residues 7-327 of the alpha 2 chain, and it appeared to be a deletion of about 30 amino acids. The pro-alpha 2(I) chains with the apparent deletion associated with normal pro-alpha 1(I) chains synthesized by the same fibroblasts and formed triple-helical type I procollagen. The presence of the altered pro-alpha 2 chains in trimers of procollagen had two consequences in terms of the physical properties of the molecule. One was to decrease the thermal stability of the protein as judged by resistance to proteolysis at 37 degrees C and by the helix to coil transition as assayed by circular dichroism. The second consequence was to make type I procollagen containing the shortened pro-alpha 2(I) chains resistant to digestion by procollagen N-proteinase. The simplest explanation for the data is that the apparent deletion in half the pro-alpha 2(I) chains produced a partial unfolding of the N-terminal region of type I procollagen which prevented processing of the protein by procollagen N-proteinase.
Osteogenesis imperfecta: cloning of a pro-alpha 2(I) collagen gene with a frameshift mutation.
Osteogenesis imperfecta (OI), a brittle-bone disorder, constitutes a major group of the inherited diseases of connective tissue. We have been studying an autosomal recessive form of OI in which the severely affected patient has inherited two abnormal pro-alpha 2(I) collagen alleles from consanguinous parents. Previously, nuclease S1 mapping was employed to localize a defect in the mRNA coding for the pro-alpha 2(I) collagen carboxyl-propeptide. The mutation prevents incorporation of pro-alpha 2(I) chains into the normal type I procollagen heterotrimer resulting in secretion of only pro-alpha 1(I) homotrimers. Here we report complete characterization of the corresponding region of the altered gene. Polyacrylamide gel electrophoresis and Southern blot hybridization showed a small homozygous deletion in the pro-alpha 2(I) collagen gene of the patient and a heterozygous pattern in both parents. Genomic cloning of the patient's DNA revealed a four nucleotide frameshift deletion in exon 1 near the end of translation which apparently instigates use of a new termination codon four nucleotides 3' to the original site. The mutation identified in this OI patient directly demonstrates the critical role of the carboxyl-propeptides in chain selection and assembly during the biosynthesis of procollagen.