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D J Prockop

Publications and source records attributed to D J Prockop.

At least 73 records · Page 4Linked to original sources
PubMed1998

Human COL9A1 and COL9A2 genes. Two genes of 90 and 15 kb code for similar polypeptides of the same collagen molecule.

Here we report the complete structure for the human COL9A1 and the complete sequence for the human COL9A2 genes. The COL9A1 gene is about 90 kb and consists of 38 exons. The COL9A2 gene is only about 15 kb, and it contains 32 exons. Sequence analysis of the promoter regions for the human COL9A2, the mouse Col9a2 and the human COL2A1 genes identified a conserved 14 bp sequence. The data also indicated that the alternative exon 1* found in intron 6 of the COL9A1 gene is separated from exon 7 only by a short intron in the chick, human, mouse and rat genes probably explaining why transcripts from exon 1* are spliced directly to exon 8.

Animals↗
PubMed1998

What holds us together? Why do some of us fall apart? What can we do about it?

One of the intriguing questions about complex organisms is, What holds them together? One of the principal answers is the tough, fibrous material known as collagen. A related question is, How is collagen made? The biosynthesis of the protein has several unusual features. One is the extensive use of the principle of spontaneous self-assembly seen in the formation of crystals. The three polypeptide chains of the protein fold into a triple-helical conformation by a process that begins with the formation of a small nucleus of triple helix at the C-terminus of the molecule and then propagation of the nucleus in a zipper-like fashion. Also, the self-assembly of the collagen monomers into fibrils is an entropy driven, crystallization-like process. Why do some of them fall apart? Mutations that alter the expression or primary structure of collagen are the predominant causes of severe skeletal defects such as osteogenesis imperfecta and chondrodysplasias. Mutations that have milder effects on the synthesis or structure of the protein are found in a subset of patients with more common diseases such as osteoporosis and early onset osteoarthritis. What can we do about the defects in collagen? Recent results have emphasized the importance of earlier observations that bone marrow contains a small subset of cells that are progenitors of osteoblasts, chondroblasts and several other types of nonhematopoietic cells. After systemic infusion into irradiated mice, the infused cells slowly replace a small fraction of the cells in bone, cartilage, lung and several other tissues. Therefore, the results suggest that the cells, known as mesenchymal stem cells or marrow stromal cells, can be used for both cell and gene therapy of diseases in which bone, cartilage and other connective tissues fall apart.

Animals↗
PubMed1998

Characterization of recombinant type II collagen: arthritogenicity and tolerogenicity in DBA/1 mice.

Recombinant human type II collagen (rhCII) was produced using both the HT1080 mammalian cell expression system (rhCIIht) and a baculovirus expression system (rhCIIbac). The biosynthesis of CII requires extensive post-translational modifications, such as the hydroxylation of prolyl and lysyl residues and glycosylation of hydroxylysyl residues. Amino acid analyses indicated that the rhCIIbac was adequately hydroxylated at prolyl residues but underhydroxylated at lysyl residues and underglycosylated compared with tissue-derived hCII, while rhCIIht was hyperhydroxylated and hyperglycosylated at lysyl residues. When the murine collagen-induced arthritis (CIA) model was used to investigate the immunological properties of the two forms of recombinant CII, each induced a high incidence of arthritis following immunization of susceptible mice when emulsified with complete Freund's adjuvant (CFA). However, the severity of the arthritis, as assessed by the number of affected limbs, was significantly higher in mice immunized with rhCIIht than in mice immunized with rhCIIbac. These data indicate that the degree of hydroxylysine glycosylation may play a role in the induction of the arthritogenic response to CII. Each of the recombinant collagens was comparable to tissue-derived hCII in their ability to induce tolerance and suppress arthritis when given as intravenous or oral tolerogens. Taken together, our data suggest that recombinant CII can be prepared in adequate amounts for therapeutic uses and that the material is immunologically comparable to tissue-derived hCII when used to induce tolerance.

Administration, Oral↗
PubMed1998

Analysis of the COL1A1 and COL1A2 genes by PCR amplification and scanning by conformation-sensitive gel electrophoresis identifies only COL1A1 mutations in 15 patients with osteogenesis imperfecta type I: identification of common sequences of null-allele mutations.

Although >90% of patients with osteogenesis imperfecta (OI) have been estimated to have mutations in the COL1A1 and COL1A2 genes for type I procollagen, mutations have been difficult to detect in all patients with the mildest forms of the disease (i.e., type I). In this study, we first searched for mutations in type I procollagen by analyses of protein and mRNA in fibroblasts from 10 patients with mild OI; no evidence of a mutation was found in 2 of the patients by the protein analyses, and no evidence of a mutation was found in 5 of the patients by the RNA analyses. We then searched for mutations in the original 10 patients and in 5 additional patients with mild OI, by analysis of genomic DNA. To assay the genomic DNA, we established a consensus sequence for the first 12 kb of the COL1A1 gene and for 30 kb of new sequences of the 38-kb COL1A2 gene. The sequences were then used to develop primers for PCR for the 103 exons and exon boundaries of the two genes. The PCR products were first scanned for heteroduplexes by conformation-sensitive gel electrophoresis, and then products containing heteroduplexes were sequenced. The results detected disease-causing mutations in 13 of the 15 patients and detected two additional probable disease-causing mutations in the remaining 2 patients. Analysis of the data developed in this study and elsewhere revealed common sequences for mutations causing null alleles.

Adult↗
PubMed1997

Apoptosis of chondrocytes in transgenic mice lacking collagen II.

Previous work demonstrated that collagen fibrils were not detectable in the cartilage of transgenic mice homozygous for targeted inactivation of the collagen II gene. In the present work, we used the same mice to show that chondrocytes undergo apoptosis in the absence of collagen II, the major component of the extracellular matrix of cartilage. The chondrocytes in the homozygous mice had condensed nuclei, fragmentation of nuclear DNA, and decreased levels of the Bcl-2 protein. These results provide direct evidence that cartilage extracellular matrix lacking collagen II cannot support the survival of chondrocytes. In addition, the results suggest that apoptosis may play a role in degenerative connective tissue diseases such as osteoarthritis in which there is extensive tissue loss.

Animals↗
PubMed1997

Marrow stromal cells as stem cells for nonhematopoietic tissues.

Marrow stromal cells can be isolated from other cells in marrow by their tendency to adhere to tissue culture plastic. The cells have many of the characteristics of stem cells for tissues that can roughly be defined as mesenchymal, because they can be differentiated in culture into osteoblasts, chondrocytes, adipocytes, and even myoblasts. Therefore, marrow stromal cells present an intriguing model for examining the differentiation of stem cells. Also, they have several characteristics that make them potentially useful for cell and gene therapy.

Adipocytes↗
PubMed1997

cDNA cloning and expression of bovine procollagen I N-proteinase: a new member of the superfamily of zinc-metalloproteinases with binding sites for cells and other matrix components.

Procollagen N-proteinase (EC 3.4.24.14) cleaves the amino-propeptides in the processing of type I and type II procollagens to collagens. Deficiencies of the enzyme cause dermatosparaxis in cattle and sheep, and they cause type VIIC Ehlers-Danlos syndrome in humans, heritable disorders characterized by accumulation of pNcollagen and severe skin fragility. Amino acid sequences for the N-proteinase were used to obtain cDNAs from bovine skin. Three overlapping cDNAs had an ORF coding for a protein of 1205 residues. Mammalian cells stably transfected with a complete cDNA secreted an active recombinant enzyme that specifically cleaved type I procollagen. The protein contained zinc-binding sequences of the clan MB of metallopeptidases that includes procollagen C-proteinase/BMP-1. The protein also contained four repeats that are homologous to domains found in thrombospondins and in properdin and that can participate in complex intermolecular interactions such as activation of latent forms of transforming growth factor beta or the binding to sulfatides. Therefore, the enzyme may play a role in development that is independent of its role in collagen biosynthesis. This hypothesis was supported by the observation that in some tissues the levels of mRNA for the enzyme are disproportionately high relative to the apparent rate of collagen biosynthesis.

Amino Acid Sequence↗
PubMed1997

Collagen II containing a Cys substitution for arg-alpha1-519. Homotrimeric monomers containing the mutation do not assemble into fibrils but alter the self-assembly of the normal protein.

A recombinant system was used to prepare human type II procollagen containing the substitution of Cys for Arg at alpha1-519 found in three unrelated families with early onset generalized osteoarthritis together with features of a mild chondrodysplasia probably best classified as spondyloepiphyseal dysplasia. In contrast to mutated procollagens containing Cys substitutions for obligatory Gly residues, the Cys substitution at alpha1-519 did not generate any intramolecular disulfide bonds. The results were consistent with computer modeling experiments that demonstrated that the alpha carbon distances were shorter with Cys substitutions for obligatory Gly residues than with Cys substitutions in the Y position residues in repeating -Gly-X-Y- sequences of the collagen triple helix. The mutated collagen did not assemble into fibrils under conditions in which the normal monomers polymerized. However, the presence of the mutated monomer in mixtures with normal collagen II increased the lag time for fibril assembly and altered the morphology of the fibrils formed.

Amino Acid Sequence↗
PubMed1997

Mass spectrometric gene diagnosis of one-base substitution from polymerase chain reaction amplified human DNA.

One-base substitution has been detected on the polymerase chain reaction (PCR) products amplified from human mutated DNA for the first time by using mass spectrometry. PCR fragments of 52 base pairs were produced on a collagen gene of an osteogenesis imperfecta patient's heterozygous DNAs. The products were digested with EcoRI restriction enzyme to liberate 3'-end adducts and purified by phenol + chloroform extraction, ammonium acetate addition and ethanol precipitation to remove sodium ions from the phosphoric acid backbone of the DNAs. Purified products were examined using an electrospray ionization mass spectrometer. Mass spectra showed four groups of fragment peaks with the expected molecular masses, which originate from the sense and antisense strands of the heterozygous DNAs.

Base Sequence↗
PubMed1997

Two new recurrent nucleotide mutations in the COL1A1 gene in four patients with osteogenesis imperfecta: about one-fifth are recurrent.

Previous observations on mutations causing osteogenesis imperfecta (OI) suggested that unrelated patients had private mutations. Here preliminary studies on two patients with type I OI indicated that some mutations in the COL1A1 gene for type I procollagen cannot be detected by analyses of cDNAs. Therefore, we developed a protocol whereby 43 exon and exon flanking sequences of the COL1A1 gene can be amplified by PCR and scanned for mutations by denaturing gradient gel electrophoresis. Two new recurrent nucleotide mutations in the gene were found in four apparently unrelated patients with OI. Analysis of previous publications indicated that up to one-fifth of the mutations causing OI are recurrent in the sense that they were identical in apparently unrelated probands. About 80% of these identical mutations were in CpG dinucleotide sequences.

Alleles↗
PubMed1997

Mutations in fibrillar collagens (types I, II, III, and XI), fibril-associated collagen (type IX), and network-forming collagen (type X) cause a spectrum of diseases of bone, cartilage, and blood vessels.

This review summarizes the data on 278 different mutations found to date in the genes for types I, II, III, IX, X, and XI collagens from 317 apparently unrelated patients. A majority (217 mutations; 78% of the total) of the mutations are single-base and either change the codon of a critical amino acid (63%), or lead to abnormal RNA splicing (13%). Most of the amino acid substitutions are those of a bulkier amino acid for the obligatory glycine of the repeating-Gly-X-Y-sequence of the collagen triple helix (155; 56%). Altogether, 26 different mutations (9.4% of the mutations) occur in more than one unrelated individual. The 65 patients in whom the 26 mutations were characterized constitute almost one-fifth (20.5%) of the 317 patients analyzed. The mutations in types I, II, III, IX, X, and XI collagens cause a wide spectrum of diseases of bone, cartilage, and blood vessels, including osteogenesis imperfecta, a variety of chondrodysplasias, types IV and VII of the Ehlers-Danlos syndrome, and, rarely, some forms of osteoporosis, osteoarthritis, and familial aneurysms.

Animals↗
PubMed1997

Synthesis and conformational properties of a recombinant C-propeptide of human type III procollagen.

A cDNA was prepared that coded for the signal peptide of type III procollagen linked to the complete C-propeptide of the protein. The cDNA was then used to express the protein in a baculovirus recombinant system. Recombinant protein was recovered as a trimer from the medium of transfected cells in a yield of 1 to 2.5 mg per liter. Mapping of peptide fragments with and without reduction indicated that the protein contained the expected interchain disulfide bonds. Analysis by circular dichroism suggested that the conformation of the protein corresponded to the native conformation. Therefore, the protein should be appropriate for further tests of its biological function and analysis of structure by X-ray diffraction.

Animals↗
PubMed1997

A recombinant homotrimer of type I procollagen that lacks the central two D-periods. The thermal stability of the triple helix is decreased by 2 to 4 degrees C.

A D-period cassette system was developed that can be used to synthesize a variety of recombinant homotrimers of type I procollagen. A construct lacking the central two D-periods of pro alpha 1(I) chains was assembled and expressed as a recombinant protein in the mammalian cell line. The recombinant protein was purified to homogeneity and the thermal stability of the triple helix assayed by rapid protease digestion. The results indicated that deletion of the central 468 amino acids from the major triple helix lowered the thermal stability of the protein by 2 to 4 degrees C. The results therefore begin to define regions of the molecule that vary in their contributions to helical stability.

Bone Morphogenetic Protein 1↗
PubMed1997

A cDNA cassette system for the synthesis of recombinant procollagens. Variants of procollagen II lacking a D-period are secreted as triple-helical monomers.

Currently there is a lack of experimental systems for defining the functional domains of the fibrillar collagens. Here we describe an experimental strategy that employs the polymerase chain reaction (PCR) to create a series of cDNA cassettes coding for seven separate domains of procollagen II. The system was used to prepare novel recombinant procollagens II from which one of the four repetitive D-periods of the triple helix was deleted. Four constructs, each lacking a different D-period, were expressed in stably transfected mammalian cells (HT-1080). Truncated procollagens of the predicted size were recovered from the medium. All were triple-helical as assayed by circular dichroism. Therefore, deletion of a complete D-period containing 234 amino acids does not destabilize the triple helix of homotrimeric collagen II as much as some naturally occurring mutations in the heterotrimeric monomer of collagen I that delete shorter sequences or that convert obligate glycine residues to residues with bulkier side chains. Moreover, the results suggest that the strategy developed here can be used to map in detail the binding sites on fibrillar collagens for other components of the extracellular matrix and for the binding, spreading and signaling of cells.

Animals↗
PubMed1997

Characterization of human type II procollagen and collagen-specific antibodies and their application to the study of human type II collagen processing and ultrastructure.

Type II collagen is the most abundant collagen in articular cartilage and, together with other tissue-specific collagens and proteoglycans, provides the tissue with its shock-absorbing properties and its resiliency to stress. Specific antibodies which recognize various collagen types have been very useful in the study of collagen biosynthesis, structure and metabolism in normal and pathological conditions. Antibodies which recognize epitopes of type II collagen have been described previously; however, many of these antibodies display cross-reactivity with other collagens or with type II collagen from other species, reflecting the high degree of homology of the helical domains of fibrillar collagens. In this study, we prepared antibodies to sequential determinants of human type II procollagen employing synthetic peptides with sequences deduced from the nucleotide sequence of the human alpha 1 (II) procollagen cDNA. The antibodies were highly specific for epitopes in either the C-terminal propeptide or the telopeptide of the human type II collagen and did not cross-react with other human interstitial collagens or with murine type II collagen. These antibodies were used in conjunction with biosynthetic labeling to study the secretion and processing of human type II procollagen and collagen in human chondrocytes in vitro. The results indicated that a lag period of about 90 min was required for the secretion of newly synthesized type II procollagen. Conversion of the secreted procollagen into fully processed alpha-chains and their deposition in the cell layer were first apparent 240 min following the initiation of biosynthetic labeling. The antibodies were also used to examine, by immunoelectron microscopy, the structure of the extracellular matrix produced by human chondrocytes maintained in long-term cultures under conditions which permit the preservation of the cartilage-specific phenotype. These highly specific antibodies provide valuable tools to study the metabolism and structure of human type II procollagen and collagen in normal and pathologic conditions.

Amino Acid Sequence↗