Synthesis of structurally unstable type III procollagen in patients with cerebral artery aneurysm

The biosynthesis of collagen was studied in skin fibroblast cultures established from 11 patients with cerebral artery aneurysms. Six patients had familial subarachnoid hemorrhage (SAH), while five patients were considered as sporadic cases. The structural stability of the triplet-helical medium procollagen was studied by measuring the thermal denaturation temperature (T_m) of type I and type II procollagen molecules. Structural instability of type III procollagen was demonstrated in two patients with familial SAH. Te T_m of type III procollagen was 39.0°C and 39.5°C in two of the cell lines, while the control value was 40.3°C. The stability of type I procollagen did not differ from that of the controls, and the main features of the biosynthesis of collagen were similar in the aneurysm patient cell lines and in the controls. The results suggest that a structural defect of type III procollagen may serve as an etiological factor in the formation of cerebral artery aneurysms.     

A disorder of collagen biosynthesis in patients with cerebral artery aneurysm

12 patients with subarachnoid hemorraghe due to rupture of a cerebral aneurysm were examined clinically for symptoms and signs of a connective tissue disorder and biochemically for details of the biosynthesis of collagen. No uniform clinical pattern of any connective tissue disorder was seen in these patients, although selected signs were observed. Skin fibroblast cultures were then established. The rate of procollagen production in two cell lines was reduced by 40% and 50%, respectively, and the intracellular accumulation of hydroxyl[¹⁴C]proline (as a percentage of total hydroxy[¹⁴C]proline) was increased by 70% in each relative to eight control cell lines. No difference was found in the degree of intracellular degradation of procollagen. After pulse-labelling, however, the radioactive procollagen was secreted into the medium in 1 h in the control cells, but required at least 3 h in the two aneursym patient cell lines. The results, thus, suggest that delayed secretion of procollagen rather than increased intracellular degradation led to the reduction in the rate of procollagen synthesis in these two fibroblast lines from patients with cerebral artery aneurysm.     

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

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

Molecular defects of type III procollagen in Ehlers-Danlos syndrome type IV

Summary Fibroblasts from most patients with Ehlers-Danlos syndrome (EDS) type IV, a disorder characterized by fragility of skin, blood vessels, and internal organs, secrete reduced amounts of type III procollagen. In 7 of 8 cell strains analyzed, we found evidence of structural defects in half of the type III procollagen chains synthesized, such as deletions or bona fide amino acid substitutions, which cause delayed formation and destabilization of the collagen triple helix and, as a consequence, reduced secretion of the molecule. The data suggest that EDS type IV is often caused by heterozygosity for mutations at the COL3A1 locus, which affect the structure of type III procollagen. The triple-helical region of the molecule, like the homologous region of type I procollagen, appears to be particularly vulnerable.Parts of this work have been presented at the 2nd International Conference on Molecular Biology and Pathology of Matrix, Philadelphia, June 15–18, 1988     

Processing of types I and III procollagen in Ehlers-Danlos syndrome type VII.

The processing of types I and III procollagen was studied in skin fibroblast cultures from type VII A and B of the Ehlers-Danlos syndrome [EDS] and age-matched controls. Synthesis of collagenous proteins was significantly increased in EDS type VII B, and the activities of prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase were slightly increased in these cell lines, reflecting increased biosynthesis of collagen. The synthesis of collagenous proteins was close to normal in EDS type VII A cells. The synthesis of type III procollagen per cell was increased, as also was the ratio of immunoreactive type III procollagen to total collagen production. The activity of type I procollagen amino-terminal proteinase was decreased in skin fibroblasts of type VII A and normal in those of type VII B relative to cell protein or DNA. Type III amino-terminal proteinase activity was of a level found in normal cells when expressed relative to the protein or DNA, and the release of type III amino-terminal propeptides was nevertheless not disturbed in these EDS type VII cell cultures. The results show that only the conversion of type I procollagen is defective in EDS type VII, and no general defect in procollagen processing can be found in EDS type VII as has been suggested in the case of dermatosparaxis, a connective tissue disorder in animals caused by disturbed procollagen conversion.     

Serum levels of type III procollagen peptide in diabetes mellitus

Serum levels of type III procollagen peptide (P-III-P) were investigated in 19 patients with type 1 (insulin-dependent) and in 48 (25 orally treated, 23 insulinized) patients with type 2 (non insulin-dependent) diabetes mellitus. Among patients with type 2 diabetes, 16 orally treated and 14 insulin-treated subjects had macrovascular complications. P-III-P levels were not correlated with the duration of diabetes and with glucose control, nor were there any significant sex and age differences in the levels. P-III-P values were significantly higher in the sera of insulin-treated non insulin-dependent diabetic patients with macroangiopathy. These high values (18.5 +/- 10.8 ng/ml) were in contrast with normal values in healthy subjects (8.5 +/- 2.5, P less than 0.001), insulin-dependent diabetics (9.9 +/- 3.4 ng/ml, P less than 0.01), non insulin-dependent diabetics treated with oral agents (8.2 +/- 2.6 ng/ml, P less than 0.001) and insulin-treated non insulin-dependent patients without macroangiopathy (8.2 +/- 4.9 ng/ml, P less than 0.001). Although this study does not demonstrate that an increase in type III collagen synthesis is responsible for the pathogenesis of macroangiopathy, it suggests that insulin-dependent fibroblast sensitization may play a role in the acceleration and progression of macroangiopathy.     

Immunochemical properties of the aminopropeptide of procollagen type III

The precursor-specific aminopropeptide of bovine type III procollagen is a strong immunogen in rabbits, guinea pigs and mice and induces antibodies which do not cross-react with type I procollagen. The antibody response is regulated by immune response genes associated with the major histocompatibility complex. Major antigenic determinants were found in the compact, non-collagenous domain (fragment Col 1) located at the N terminus of the aminopropeptide and were destroyed by reduction of disulfide bonds. Minor antigenic determinants independent of disulfide bonds also exist in fragment Col 1 and could be localized on a distinct tryptic peptide. Fragment Col 1 showed a lower affinity for antibody when compared with the intact aminopropeptide which causes a non-parallel shift in radioimmuno-inhibition profiles. Monovalent antibody fragments showed an average tenfold reduction in affinity constant and failed to distinguish between aminopropeptide and fragment Col 1. This indicates that the stronger binding of bivalent antibody by the triple-stranded aminopropeptide is due to multiple interactions with both antibody binding sites which are lost for a single-stranded antigen (Col 1) or with monovalent antibody fragments.     

Transgenic silkworms produce recombinant human type III procollagen in cocoons

We describe the generation of transgenic silkworms that produce cocoons containing recombinant human collagen. A fusion cDNA was constructed encoding a protein that incorporated a human type III procollagen mini-chain with C-propeptide deleted, a fibroin light chain (L-chain), and an enhanced green fluorescent protein (EGFP). This cDNA was ligated downstream of the fibroin L-chain promoter and inserted into a piggyBac vector. Silkworm eggs were injected with the vectors, producing worms displaying EGFP fluorescence in their silk glands. The cocoons emitted EGFP fluorescence, indicating that the promoter and fibroin L-chain cDNAs directed the synthesized products to be secreted into cocoons. The presence of fusion proteins in cocoons was demonstrated by immunoblotting, collagenase-sensitivity tests, and amino acid sequencing. The fusion proteins from cocoons were purified to a single electrophoretic band. This study demonstrates the viability of transgenic silkworms as a tool for producing useful proteins in bulk.     

Purification and characterization of the N-terminal propeptide of human type III procollagen.

The N-terminal propeptide of type III procollagen was purified from human ascitic fluid by using (NH4)2SO4 precipitation, DEAE-Sephacel chromatography at pH 8.6, Sephacryl S-300 chromatography and another DEAE-Sephacel chromatography at pH 4.5. The Mr of the human peptide was about 42 000, which corresponds in size to the propeptide released by the specific N-proteinase during the extracellular processing of collagen. Bacterial-collagenase digestion of the human peptide produced three fragments, which could be separated on a Bio-Gel P-10 column. The human propeptide and its collagenase-derived fragments, an N-terminal non-collagenous domain Col 1, a C-terminal non-helical domain Col 2 and a collagenous domain Col 3, resembled those derived from the N-terminal segment of bovine type III procollagen in their amino acid composition. The human peptide was found to contain sulphate, which may explain its extremely low isoelectric point (3.1). Antibodies against the human N-terminal propeptide reacted similarly with both the purified human peptide and a corresponding segment of bovine type III procollagen. The human propeptide could be used in developing radioimmunoassays for monitoring fibrotic processes.     

Activities of enzymes of collagen biosynthesis and levels of type III procollagen peptide in the serum of patients with sarcoidosis

Serum immunoreactive prolylhydroxylase (IRPH), galactosylhydroxylsyl glucosyltransferase activity (GGT) and amino-terminal propeptides of type III procollagen (Pro(III) peptide) were measured in fifty three patients with sarcoidosis (all having some degrees of pulmonary fibrosis). The levels of IRPH and Pro(III) peptide showed no relationships to the clinical assessment of the disease and while GGT activity was raised in approximately 80% of the patients there was no correlation between the size of the increases and the clinical activity of the disease. The results of this study would suggest that measurement of the above parameters offer no specificity in either diagnosing or assessing the clinical activity of sarcoidosis. The observed increases in serum GGT activity in affected patients would however suggest that measurement of this enzyme may be useful perhaps in more severe pulmonary fibrotic reactions.     

Purification of human procollagen type III N-proteinase from placenta and preparation of antiserum.

Procollagen type III N-proteinase, of Mr about 70,000, was detected in human placental tissue and purified from this source more than 5800-fold. It was found to be a glycoprotein, which was bound to both concanavalin A-Ultrogel and heparin-Sepharose affinity columns. Binding to a type III pN-collagen-Sepharose affinity column was used as the final step in purification. The purified enzyme accepted only native type III procollagen or [14C]carboxymethylated type III pN-collagen as its substrate; type I, type II and type IV procollagen and heat-denatured type III pN-collagen were not cleaved by the enzyme. Antibodies against this purified enzyme protein raised in rabbits demonstrated a high inhibitory effect on the enzyme activity. Immunoblotting of the denatured protein and immunoelectrophoresis of the native enzyme showed only one major antigenic component, again with an Mr of about 70,000. The antibodies cross-reacted with the enzyme preparation from foetal-calf aorta smooth-muscle cells.     

Immunofluorescent localization of type III collagen and the N-terminal propeptide of type III procollagen in dentin matrix in osteogenesis imperfecta

Demineralized deciduous and permanent teeth from seven patients with six different types of osteogenesis imperfecta (OI) and from four unaffected controls were stained for type III collagen and for the N-terminal propeptide of type III procollagen using indirect immunofluorescence. Sillence types IA, IB and III OI were each represented by one patient. Two patients had type IVB and two had unclassifiable OI. After enzymatic treatment, the dentin matrix of one patient each with type IB OI, type IVB, and unclassifiable OI reacted with the specific antibodies against both type III collagen and the N-terminal propeptide. Positive staining was observed around the pathological canal-like structures and as delicate strands traversing the matrix. The similar patterns of immunofluorescence for both antigens in dentin in OI are suggestive of retention of the N-terminal propeptide in association with type III collagen identical to that in normal nonmineralized connective tissues. The abnormal presence of type III collagen in dentin in OI may be secondary to the aberrant structure of type I collagen. The failure of dentin matrix of all patients with OI to immunostain for type III collagen and the N-terminal propeptide may reflect heterogeneity or additional secondary changes in matrix macromolecule interactions.     

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

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

Structure of cDNA clones coding for the entire prepro alpha 1 (III) chain of human type III procollagen. Differences in protein structure from type I procollagen and conservation of codon preferences.

Two overlapping cDNA clones that cover the complete length of the mRNA for human type III procollagen were characterized. The data provided about 2500 base pairs of sequence not previously defined for human type III procollagen. Two tripeptide sequences of -Gly-Xaa-Yaa- were identified that were not detected previously by amino acid sequencing of human type III collagen. The two additional tripeptide units, together with three previously detected, establish that the alpha 1 (III) chain is 15 amino acids longer than either the alpha 1 (I) or alpha 2 (I) chains of type I collagen. The additional tripeptide units made hydropathy plots of the N-terminal and C-terminal regions of type III collagen distinctly different from those of type I collagen. The data also demonstrated that human type III procollagen has the same third base preference in codons for glycine, proline and alanine that was previously found with human and chick type I procollagen. In addition, comparison of two cDNA clones from the same individual revealed a variation in structure in that the codon for amino acid 880 of the alpha 1 (III) chain was -CTT- for leucine in one clone and -TTT- for phenylalanine in the other.     

Development and characterization of monoclonal antibodies to human type III procollagen

Hybridomas which secrete monoclonal antibodies against human type III procollagen have been developed. By an enzyme-linked immunosorbent assay, three of the monoclonal antibodies have been determined to be against non-helical extensions of the molecules while two of the antibodies are against helical portion of the molecules which is sensitive to bacterial collagenase action. These findings have been further confirmed by carrying out immuno-reaction of the pro α-chains transferred on nitrocellulose paper from sodium dodecyl sulfate polyacrylamide gels. These monoclonal antibodies have been found to be suitable reagents for immunohistochemical studies as well as for immunoassays of type III procollagen and collagen.     

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.     

Complete amino acid sequence of the N-terminal extension of calf skin type III procollagen.

The N-terminal extension peptide of type III procollagen, isolated from foetal-calf skin, contains 130 amino acid residues. To determine its amino acid sequence, the peptide was reduced and carboxymethylated or aminoethylated and fragmented with trypsin, Staphylococcus aureus V8 proteinase and bacterial collagenase. Pyroglutamate aminopeptidase was used to deblock the N-terminal collagenase fragment to enable amino acid sequencing. The type III collagen extension peptide is homologous to that of the alpha 1 chain of type I procollagen with respect to a three-domain structure. The N-terminal 79 amino acids, which contain ten of the 12 cysteine residues, form a compact globular domain. The next 39 amino acids are in a collagenase triplet sequence (Gly- Xaa - Yaa )n with a high hydroxyproline content. Finally, another short non-collagenous domain of 12 amino acids ends at the cleavage site for procollagen aminopeptidase, which cleaves a proline-glutamine bond. In contrast with type I procollagen, the type III procollagen extension peptides contain interchain disulphide bridges located at the C-terminus of the triple-helical domain.