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The Journal of Biological Chemistry Aug 1991Expression in a recombinant system has been difficult to obtain for any of the major fibrillar collagens that require processing by eight or more post-translational...
Expression in a recombinant system has been difficult to obtain for any of the major fibrillar collagens that require processing by eight or more post-translational enzymes. Here, two DNA constructs were designed so that the promoter region of the gene for the pro-alpha 1(I) chain of human type I procollagen drove expression of the human type II procollagen gene in mouse NIH 3T3 cells, a culture line that normally synthesizes type I procollagen but not any cartilage-specific protein such as type II procollagen. Both constructs were expressed as both mRNA and protein. In clones expressing the construct at high levels, the steady-state levels of mRNA and the production of type II procollagen were comparable to the mRNA levels and production of type I procollagen from the endogenous mouse genes. Comparison of clones containing the two constructs demonstrated that sequences extending 80 base pairs beyond the major polyadenylation signal of the gene are not in themselves sufficient for correct termination and 3' processing of RNA transcripts. The results strongly suggest that specific sequences present in a downstream 3.5-kilobase SphI/SphI fragment determine the termination of the transcription. Of special importance is that the system will make it possible to examine the consequences of mutations in the human type II procollagen gene on the processing of RNA transcripts and on the functional properties of the protein simply by using the genomic DNA from leukocytes or other non-cartilaginous sources.
Topics: Animals; Base Sequence; Blotting, Northern; Blotting, Western; Cloning, Molecular; Cyanogen Bromide; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation; Humans; Mice; Plasmids; Procollagen; RNA, Messenger; Transcription, Genetic; Transfection
PubMed: 1860834
DOI: No ID Found -
The American Review of Respiratory... Apr 1986Pulmonary basement membranes are believed to play important roles in lung morphogenesis, in maintenance of lung alveolar architecture, and in repair after pulmonary...
Pulmonary basement membranes are believed to play important roles in lung morphogenesis, in maintenance of lung alveolar architecture, and in repair after pulmonary injury. However, very little is known about the synthesis and matrix deposition of lung basement membrane macromolecules. Accordingly, we have investigated the synthesis of type IV procollagen, the major collagenous component of basement membranes, in slices of adult rat lung. After a 4-h labeling with [3H]proline, type IV procollagen chains were extracted from lung homogenates with 2 M guanidine-HCl and purified by salt fractionation and ion exchange chromatography. The chains comigrated with authentic type IV chains by SDS-PAGE and were selectively coprecipitated with antibodies to type IV collagen. Peptide mapping confirmed their identity as pro alpha 1(IV) and pro alpha 2(IV), and suggested that the chains are predominantly assembled as heteropolymers. There was no evidence for proteolytic processing of the newly synthesized type IV procollagen even though type I and III procollagens were rapidly processed to lower molecular weight intermediates and collagen. Type IV procollagen accounted for approximately 40% of the extracted radiolabeled collagen, suggesting that there may be a relatively high turnover of lung basement membrane collagen in vivo.
Topics: Animals; Electrophoresis, Polyacrylamide Gel; Guanidine; Guanidines; Lung; Male; Organ Culture Techniques; Procollagen; Proline; Rats; Rats, Inbred Strains; Tritium
PubMed: 3963627
DOI: 10.1164/arrd.1986.133.4.618 -
The EMBO Journal 1982Type IV procollagen was isolated from the culture medium of the teratocarcinoma cell line PYS-2 by affinity chromatography on heparin-Sepharose. Immunological studies...
Type IV procollagen was isolated from the culture medium of the teratocarcinoma cell line PYS-2 by affinity chromatography on heparin-Sepharose. Immunological studies showed that type IV procollagen is composed of pro-alpha 1(IV) and pro-alpha 2(IV) chains and contains two potential cross-linking sites which are located in the short triple-helical 7S domain and the globular domain NC1 . The 7S domain was also identified as the heparin binding site. Rotary shadowing visualized type IV procollagen as a single triple-helical rod (length 388 nm) with a globule at one end. Some of the procollagen in the medium, however, had formed aggregates by alignment of 2-4 molecules along their 7S domains. After deposition in the cell matrix, non-reducible cross-links between the 7S domains are formed while the globules of two procollagen molecules connect to each other. The latter may require a slight proteolytic processing of the globular domains NC1 . The shape of type IV procollagen and the initial steps in its assembly are compatible with a recently proposed network of type IV collagen molecules in basement membranes. Since both type IV collagen and laminin bind to heparin, the formation of higher ordered structures by interaction of both proteins with heparan-sulfate proteoglycan may occur in situ.
Topics: Animals; Cell Line; Electrophoresis, Polyacrylamide Gel; Macromolecular Substances; Microscopy, Electron; Procollagen; Protein Conformation; Teratoma
PubMed: 7188361
DOI: 10.1002/j.1460-2075.1982.tb01251.x -
The Biochemical Journal Feb 1994Apparently because the biosynthetic pathways involve eight or more highly specific post-translational enzymes, it has been difficult to obtain expression of genes for...
Apparently because the biosynthetic pathways involve eight or more highly specific post-translational enzymes, it has been difficult to obtain expression of genes for fibrillar collagens in recombinant systems. Here two constructs of the human gene for procollagen II (COL2A1) were prepared, one with about 0.5 kb of a promoter for a procollagen I gene (COL1A1) and the other with about 4 kb of the promoter for the procollagen II gene. The constructs, together with a neomycin-resistant gene, were transfected into a human tumour cell line (HT1080) that synthesizes the collagen IV found in basement membranes, but does not synthesize any fibrillar collagen. About two per 100 clones resistant to the neomycin analogue G418 synthesized and secreted human procollagen II. Milligram quantities of the recombinant procollagen II were readily isolated from the cultured medium. The recombinant procollagen II had the expected amino acid sequence as defined by nucleotide sequencing of mRNA-derived cDNA and the expected amino acid composition as defined by analysis of procollagen II that was converted into collagen II by digestion with procollagen N- and C-proteinases. Also, analysis of the carbohydrate content indicated that there was glycosylation of some of the hydroxylysine residues but no evidence of post-translational overmodification of the residues. In addition, the protein was shown to have a native conformation as assayed by a series of protease digestions. No essential differences were found between clones transfected with the COL2A1 gene construct containing the COL1A1 promoter and the similar construct containing the COL2A1 promoter in terms of number of clones synthesizing recombinant procollagen II and the levels of expression. With both constructs, the expression of the COL2A1 gene was closely related to copy number. The results demonstrated therefore that it is not essential to use a promoter for a gene normally expressed in a host cell in order to obtain gene copy-number-dependent expression of an exogenous collagen gene in stably transfected cells.
Topics: Amino Acid Sequence; Amino Acids; Chromatography, DEAE-Cellulose; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Humans; Molecular Sequence Data; Procollagen; Promoter Regions, Genetic; Recombinant Proteins; Transfection; Tumor Cells, Cultured
PubMed: 8129728
DOI: 10.1042/bj2980031 -
Journal of Cellular Biochemistry 1985Conversion from procollagen to collagen is a specific process that is a requirement for proper alignment of collagen molecules to form functional fibers. This process is... (Review)
Review
Conversion from procollagen to collagen is a specific process that is a requirement for proper alignment of collagen molecules to form functional fibers. This process is catalyzed by at least three structurally and functionally distinct enzymes cleaving collagen types I-III. The cleavage processes possibly taking place in the more recently discovered collagen types are not known to any extent at this time. Two amino-terminal proteinases, one cleaving type I and type II procollagens and the other cleaving type III procollagen, have been purified close to homogeneity, and the more unspecific activity of carboxy-terminal proteinase has been isolated from several tissues. In our experimental model, however, cleavage of the carboxy-terminal propeptides of types I and III procollagen is differently affected by lysine. This suggests the presence of at least two distinct enzymes for the removal of carboxyl-terminal propeptides. The regulation of the reaction process from procollagen to collagen is not well known at present. The importance of the phenomenon in terms of fibril formation, however, is demonstrated by several elegant studies in vitro; and certain genetic disorders in which this process is defective demonstrate the significance in vivo. Moreover, the factors shown to effect the cleavage process may be potentially beneficial in the treatment of the pathological processes with abnormal collagen accumulation such as fibrosis. In this paper we briefly review the current knowledge of the converting enzymes, including some very recent findings of our laboratory as well as the evidence presented for the biological significance of the conversion process.
Topics: Animals; Bone Morphogenetic Protein 1; Bone Morphogenetic Proteins; Cattle; Chick Embryo; Collagen; Ehlers-Danlos Syndrome; Endopeptidases; Humans; Lysine; Metalloendopeptidases; Mice; Osteogenesis Imperfecta; Procollagen; Procollagen N-Endopeptidase; Protease Inhibitors; Protein Processing, Post-Translational
PubMed: 3928635
DOI: 10.1002/jcb.240280104 -
The Journal of Biological Chemistry Dec 1985Procollagen carboxyl-terminal proteinase, the enzyme which cleaves the carboxyl-terminal propeptides from type I procollagen, was extensively purified in a yield of 25%...
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.
Topics: Amino Acid Sequence; Animals; Bone Morphogenetic Protein 1; Bone Morphogenetic Proteins; Calcium; Chick Embryo; Chromatography, Affinity; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Kinetics; Metalloendopeptidases; Molecular Weight; Procollagen; Tendons
PubMed: 3905801
DOI: No ID Found -
Biochemistry Aug 1978Type I procollagen mRNAs were separated from contaminating low-abundance messenger and nuclear RNAs by chromatography over Sepharose 4B in 0.65 M NaCl at room...
Type I procollagen mRNAs were separated from contaminating low-abundance messenger and nuclear RNAs by chromatography over Sepharose 4B in 0.65 M NaCl at room temperature. All of 27S rRNA and four-fifths of procollagen mRNAs bind to Sepharose under these conditions, while 18S rRNA and about three-fourths of other poly(A)-containing RNAs do not bind. AMV reverse transcriptase was used to prepare complementary DNA to procollagen mRNA at each purification step. Hybridization studies, in RNA excess, were carried out to establish the enrichment at each step both with respect to total RNA and to poly(A)-containing RNA. While "purified" procollagen mRNA preparations still consist of about 50% 27S rRNA, over 80% of cDNA prepared from it back hybridizes to its template at a log of cr0t1/2 of -1.9. This type I procollagen cDNA hybridizes in DNA excess to DNA isolated from chicken erythrocytes and from embryonic chick calvaria at a log c0t1/2 of 3.1, demonstrating that procollagen cDNA is complementary to unique gene sequences in both tissues and that procollagen genes are not reiterated.
Topics: Animals; Bone and Bones; Chick Embryo; Chickens; DNA; Genes; Kinetics; Molecular Weight; Nucleic Acid Hybridization; Nucleic Acid Renaturation; Procollagen; RNA, Messenger; Reticulocytes
PubMed: 687581
DOI: 10.1021/bi00609a011 -
Experimental Cell Research Apr 2001To investigate the molecular mechanism of intracellular degradation of type I collagen in normal corneal endothelial cells (CEC), we studied the role of prolyl...
To investigate the molecular mechanism of intracellular degradation of type I collagen in normal corneal endothelial cells (CEC), we studied the role of prolyl 4-hydroxylase (P4-H) and protein disulfide-isomerase (PDI; the beta subunit of P4-H) during procollagen I biosynthesis. When the subcellular localization of P4-H and PDI was determined, P4-H demonstrated a characteristic diffuse endoplasmic reticulum (ER) pattern, whereas PDI showed a slightly more restricted distribution within the ER. When colocalization of procollagen I with the enzymes was examined, procollagen I and PDI showed a large degree of colocalization. P4-H and procollagen I were predominantly colocalized at the perinuclear site. When colocalization of type IV collagen with PDI and P4-H was examined, type IV collagen was largely colocalized with PDI, which showed a wider distribution than type IV collagen. Type IV collagen is similarly colocalized with P4-H, except in some perinuclear sites. The colocalization profiles of procollagen I with both PDI and P4-H were not altered in cells treated with alpha,alpha'-dipyridyl compared to those of the untreated cells. The underhydroxylated type IV collagen demonstrated a colocalization profile with PDI similar to that observed with procollagen I, while the underhydroxylated type IV collagen was predominantly colocalized with P4-H at the perinuclear sites. Immunoblot analysis showed no real differences in the amounts of the beta subunit/PDI and the catalytic alpha subunit of P4-H in CEC compared to those of corneal stromal fibroblasts (CSF). When protein-protein association was determined, procollagen I was associated with PDI much more in CEC than it was in CSF, whereas type IV collagen showed no differential association specificity to PDI in both cells. Limited proteolysis of the newly synthesized intracellular procollagen I with pepsin showed that procollagen I in CEC was degraded by pepsin, whereas CSF contained type I collagen composed of alpha1(I) and alpha2(I). These findings suggest that procollagen I synthesized in CEC is not in triple helical conformation and that the improperly folded procollagen I may be preferentially associated with PDI before targeting to the intracellular degradation.
Topics: Animals; Cells, Cultured; Endothelium, Corneal; Procollagen; Procollagen-Proline Dioxygenase; Protein Conformation; Protein Disulfide-Isomerases; Rabbits; Subcellular Fractions
PubMed: 11262193
DOI: 10.1006/excr.2000.5155 -
Cell Feb 1977Procollagen and collagen were isolated from the culture medium and cell layer of line TSD4 (obtained from mouse teratocarcinoma OTT6050). SDS-polyacrylamide gel...
Procollagen and collagen were isolated from the culture medium and cell layer of line TSD4 (obtained from mouse teratocarcinoma OTT6050). SDS-polyacrylamide gel electrophoresis of the highly purified procollagen fraction demonstrated that the fraction is composed of theta chains (150,000 daltons), pro alpha chains (130,000 daltons), and alpha chains (100,000 daltons). Limited pepsin digestion of this fraction yielded a single species of collagen molecules having a chain composition (alpha1)3, as did collagen isolated from the cell layer. Each alpha1 chain appears to be slightly larger than alpha1 chains from calf or human type I and type III collagen. Amino acid analysis and cyanogen bromide peptide profiles of pepsin-treated TSD4 collagen demonstrated significant differences from those of other collagens (II, III, IV) of the type alpha1(X)3, although similar to that of the alpha1 chain of type I collagen, [alpha1(1)]2alpha2. Taken together, acrylamide gel electrophoresis, amino acid composition, electron microscopy, and cyanogen bromide peptide analysis indicate that this material represents a new molecular species of collagen not previously characterized, probably related to [alpha1(I)]3.
Topics: Amino Acids; Cell Line; Collagen; Electrophoresis, Polyacrylamide Gel; Microscopy, Electron; Molecular Weight; Peptides; Procollagen; Protein Conformation; Teratoma
PubMed: 837450
DOI: 10.1016/0092-8674(77)90222-7 -
The Journal of Biological Chemistry Oct 2010Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs)...
Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs) such as bone morphogenetic protein-1 (BMP-1). The PCPs can process additional extracellular protein precursors and play fundamental roles in developmental processes and assembly of the ECM. The stimulatory activity of PCPE-1 is restricted to the processing of fibrillar procollagens, suggesting PCPE-1 is a specific regulator of collagen deposition. PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptides and are required for PCP enhancing activity, and one NTR domain that binds heparin. To understand the biological role of the NTR domain, we performed surface plasmon resonance (SPR) binding assays, cell attachment assays as well as immunofluorescence and activity assays, all indicating that the NTR domain can mediate PCPE-1 binding to cell surface heparan sulfate proteoglycans (HSPGs). The SPR data revealed binding affinities to heparin/HSPGs in the high nanomolar range and dependence on calcium. Both 3T3 mouse fibroblasts and human embryonic kidney cells (HEK-293) attached to PCPE-1, an interaction that was inhibited by heparin. Cell attachment was also inhibited by an NTR-specific antibody and the NTR fragment. Immunofluorescence analysis revealed that PCPE-Flag binds to mouse fibroblasts and heparin competes for this binding. Cell-associated PCPE-Flag stimulated procollagen processing by BMP-1 several fold. Our data suggest that through interaction with cell surface HSPGs, the NTR domain can anchor PCPE-1 to the cell membrane, permitting pericellular enhancement of PCP activity. This points to the cell surface as a physiological site of PCPE-1 action.
Topics: 3T3 Cells; Animals; Cell Line; Collagen; Extracellular Matrix Proteins; Glycoproteins; Heparin; Heparitin Sulfate; Humans; Kinetics; Mice; Microscopy, Fluorescence; Procollagen; Protein Binding; Protein Structure, Tertiary
PubMed: 20729553
DOI: 10.1074/jbc.M110.141366