Surface plasmon resonance detection of interactions between peptide fragments of N-telopeptide and its monoclonal antibodies.

As populations age, osteoporosis is becoming an important public health care problem. Urinary level of the cross-linked N-telopeptide of type I collagen has been reported to be a sensitive marker of bone resorption. Recently, we synthesized and characterized 10 overlapping peptides covering the N-telopeptide of alpha-2 type I collagen and reported their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibodies determined by a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). In this study, we design an assay based on the surface plasmon resonance (SPR) technology to detect binding interaction of each peptide fragment of NTX with the anti-NTX monoclonal antibodies. Anti-NTX monoclonal antibodies were immobilized on the surface of sensor chip by amine-coupling procedure. Serial dilutions of each peptide were prepared and injected separately onto the antibodies-immobilized sensor chip. The real-time association and dissociation interactions of each peptide were detected and reported as sensorgrams. Binding response of each peptide to the monoclonal antibodies was determined, and the SPR results were compared with the ELISA results. We demonstrate that the trends of binding potency of peptide fragments detected by SPR are in good correlation to the results obtained by ELISA, indicating that our developed SPR-based method can be further applied to detect the NTX fragments in urine and to monitor the bone loss in humans. The potent peptide fragments identified by both assays are promising for further preparation of specific monoclonal antibodies in order to develop bioassays for bone loss in humans.     

A cross-sectional study on biochemical parameters of bone turnover and vitamin d metabolites in healthy dutch children and young adults

AIM: To provide reference data of biochemical markers of bone turnover and vitamin D metabolites for children and young adults. METHODS: Blood samples were taken from 176 healthy Dutch children and young adults (age range 7.6-25.3 years) to assess serum calcium, alkaline phosphatase, inorganic phosphate, osteocalcin, collagen type I cross-linked N-telopeptide, N-terminal propeptide of type I procollagen, 25-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 levels. Cross-linked telopeptide of type I collagen and carboxy-terminal propeptide of type I procollagen were assessed in 286 subjects (age range 1.4-25.3 years). RESULTS: Calcium and vitamin D levels were independent of age. The peak concentrations for collagen type I cross-linked N-telopeptide, cross-linked telopeptide of type I collagen, carboxy-terminal propeptide of type I procollagen, N-terminal propeptide of type I procollagen, alkaline phosphatase, and osteocalcin were found during puberty, in girls approximately 2.5 years earlier than in boys. Strong correlations were found between the markers of bone turnover, while no correlation was found between the markers of bone turnover and bone mineral density measured by dual-energy X-ray absorptiometry. CONCLUSIONS: Single measurements of bone markers cannot predict bone density. Reference data according to gender, age, and Tanner stage are given which allow calculating standard deviation scores adjusted for age and gender.     

Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro

During bone and dentin mineralization, the crystal nucleation and growth processes are considered to be matrix regulated. Osteoblasts and odontoblasts synthesize a polymeric collagenous matrix, which forms a template for apatite initiation and elongation. Coordinated and controlled reaction between type I collagen and bone/dentin-specific noncollagenous proteins are necessary for well defined biogenic crystal formation. However, the process by which collagen surfaces become mineralized is not understood. Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein expressed during the initial stages of mineralized matrix formation in bone and dentin. Here we show that DMP1 bound specifically to type I collagen, with the binding region located at the N-telopeptide region of type I collagen. Peptide mapping identified two acidic clusters in DMP1 responsible for interacting with type I collagen. The collagen binding property of these domains was further confirmed by site-directed mutagenesis. Transmission electron microscopy analyses have localized DMP1 in the gap region of the collagen fibrils. Fibrillogenesis assays further demonstrated that DMP1 accelerated the assembly of the collagen fibrils in vitro and also increased the diameter of the reconstituted collagen fibrils. In vitro mineralization studies in the presence of calcium and phosphate ions demonstrated apatite deposition only at the collagen-bound DMP1 sites. Thus specific binding of DMP1 and possibly other noncollagenous proteins on the collagen fibril might be a key step in collagen matrix organization and mineralization.     

Separate and overlapping specificities in rheumatoid arthritis antibodies binding to citrulline- and homocitrulline-containing peptides related to type I and II collagen telopeptides

IntroductionOur objective was to find out if there are antibodies binding to homocitrulline-containing type I and II collagen carboxyterminal telopeptides in sera of patients with rheumatoid arthritis (RA), and if these antibodies cross-react with citrulline and homocitrulline in the same peptide sequence.MethodsA total of 72 RA and 72 control sera were analyzed for binding using enzyme-linked immunosorbent assay to citrulline- or homocitrulline-containing type I and II collagen carboxyterminal telopeptides, as well as to cyclic citrullinated peptide (CCP) and to mutated citrullinated vimentin (MCV). Specificities of the antibodies were tested using inhibition-ELISA.ResultsOf the RA sera, 39 (54%) and 41 (57%) were positive for binding to CCP and MCV, respectively. Further, 34 (47%) and 30 (42%) of the patients had specific antibodies binding to and being inhibited by citrulline-containing type I collagen telopeptides and by citrulline-containing type II collagen carboxyterminal telopeptides, respectively. The corresponding figures regarding homocitrulline-containing type I and homocitrulline-containing type II collagen telopeptides were 16 (22%) and 14 (19%). Most of the patients, who were seropositive for citrullinated peptides, showed binding in multiple assays. A total of 10 (14%) RA patients were positive for all the tested peptide pairs, while 28 (39%) of them had antibodies that contained overlapping specifities between citrulline and homocitrulline in the same peptide sequence.ConclusionsAntibodies to both citrulline and homocitrulline containing type I and II collagen telopeptides can be found in sera of RA patients. These antibodies are not constant from one RA patient to another, but contain separate or overlapping specificities within the same peptide sequence varying between individuals. Our results suggest some relationship between citrulline and homocitrulline-recognizing antibodies, since homocitrulline antibodies exist mainly in individuals seropositive to anti-CCP and anti-MCV.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0515-z) contains supplementary material, which is available to authorized users.     

Human and chick alpha 2(I) collagen mRNA: comparison of the 5' end in osteoblasts and fibroblasts

Type I collagen, a heterotrimer of two alpha 1(I) chains and one alpha 2(I) chain, is the major structural protein of bone, skin, and tendon. The collagen of patients with bone diseases has been studied in skin fibroblasts instead of osteoblasts because the genes for type I collagen are single-copy genes. While these studies should detect structural changes in the gene, they might fail to detect defects in processes which are dependent on tissue-specific expression. The studies reported here sought to determine whether the expression of type I collagen in skin and bone was characterized by the use of alternate promoters or alternative splicing in the N-propeptide region. Primer extension and nuclease S1 protection experiments were used to analyze the structure of the alpha 2(I) mRNA from the 5' end of the gene through the N-telopeptide coding region (exons 1-6) in human and chick osteoblasts and fibroblasts. The protection and primer extension experiments using human and chick mRNA demonstrated identical routes of splicing in skin and bone at the first five splice junctions. These studies provide reassurance that information obtained from the study of type I collagen in fibroblasts may be extrapolated to bone.     

Mapping Hsp47 binding site(s) using CNBr peptides derived from type I and type II collagen

As a crucial molecular chaperone in collagen biosynthesis, Hsp47 interacts with the nascent form as well as the mature triple-helical form of procollagen. The location(s) of Hsp47 binding sites on the collagen molecule are, as yet, unknown. We have examined the substrate specificity of Hsp47 in vitro using well-characterized CNBr peptide fragments of type I and type II collagen along with radiolabeled, recombinant Hsp47. Interaction of these peptides with Hsp47 bound to collagen-coated microtiter wells showed several binding sites for Hsp47 along the length of the alpha1 and alpha2 chains of type I collagen and the alpha1 chain of type II collagen, with the N-terminal regions showing the strongest affinities. The latter observation was also supported by the results of a ligand-blot assay. Except for two peptides in the alpha2(I) chain, peptides that showed substantial binding to Hsp47 did so in their triple-helical and not random-coil form. Unlike earlier studies that used peptide models for collagen, the results obtained here on fragments of type I and type II collagen identify, for the first time, binding of Hsp47 to specific regions of the collagen molecule. They also point to additional structural requirements for Hsp47 binding besides the known preference for third-position Arg residues and the triple-helical conformation.     

Type I collagen N-telopeptides adopt an ordered structure when docked to their helix receptor during fibrillogenesis

The in vitro rate and specificity of fibrillogenesis in type I collagen depends on the integrity of the amino (N)-telopeptide domain. In vivo an intact N-telopeptide domain is also required for normal fibril assembly. Although Chou-Fasman predictions and NMR studies suggested that a type I beta-turn could be induced in alpha1(I) N-telopeptide chains, computer modeling did not identify ordered structures. Nevertheless, X-ray analysis and electron tomography studies have shown that the N-telopeptide is in one of the most highly ordered fibril domains. This study was undertaken to determine if the docking of the N-telopeptide to its helix receptor domain could induce the telopeptides to take up a specific conformation. With use of molecular modeling suite of programs, a (Gly-Pro-Pro)(n) triple-helical structure was built on the basis of high-resolution X-ray crystallographic coordinates and then replaced with the actual bovine collagen residues 924-938, the triple-helical alpha1(I)-N-telopeptide-receptor sequences. Energy minimization produced a modified triple-helical conformation. The bovine alpha1(I) N-telopeptide sequence was similarly minimized and docked to this receptor. The docking induced an ordered conformation with a stabilizing hydrogen bond in the N-telopeptide and, importantly, a reciprocal reordering of the triple-helical conformation in the binding domain. This docked structure placed Lys residues in both telopeptide and helix in the correct locations for cross-link formation. The modeling has been extended to the three-chain N-telopeptide domain and finally to the construction of the Hulmes-Miller quasi-hexagonal packing structure. Each N-telopeptide domain can form linkages with two adjacent, aligned helix receptor domains. The telopeptides and the order of staggering of the three chains in the helix play crucial roles in the packing and intrafibrillar cross-linking patterns and the relative azimuthal orientations of adjacent molecules in the fibril. The models confirm the high order in the N-telopeptide 4D overlap zone.     

A novel binding site in collagen type III for integrins alpha1beta1 and alpha2beta1

Previously identified high affinity integrin-binding motifs in collagens, GFOGER and GLOGER, are not present in type III collagen. Here, we first characterized the binding of recombinant I domains from integrins alpha(1) and alpha(2) (alpha(1)I and alpha(2)I) to fibrillar collagen types I-III and showed that each I domain bound to the three types of collagens with similar affinities. Using rotary shadowing followed by electron microscopy, we identified a high affinity binding region in human type III collagen recognized by alpha(1)I and alpha(2)I. Examination of the region revealed the presence of two sequences that contain the critical GER motif, GROGER and GAOGER. Collagen-like peptides containing these two motifs were synthesized, and their triple helical nature was confirmed by circular dichroism spectroscopy. Experiments show that the GROGER-containing peptide was able to bind both alpha(1)I and alpha(2)I with high affinity and effectively inhibit the binding of alpha(1)I and alpha(2)I to type III and I collagens, whereas the GAOGER-containing peptide was considerably less effective. Furthermore, the GROGER-containing peptide supported adhesion of human lung fibroblast cells when coated on a culture dish. Thus, we have identified a novel high affinity binding sequence for the collagen-binding integrin I domains.     

Identification of cross-linking sites in bovine cartilage type IX collagen reveals an antiparallel type II-type IX molecular relationship and type IX to type IX bonding.

Type IX collagen functions in covalent cross-linkage to type II collagen in cartilage (Eyre, D. R., Apone, S., Wu, J. J., Ericsson, L. H., and Walsh, K. A. (1987) FEBS Lett. 220, 337-341). To understand this molecular relationship better, an analysis of all cross-linking sites labeled by [3H]borohydride was undertaken using the protein prepared from fetal bovine cartilage. Sequence analysis of tryptic peptides containing the 3H-labeled cross-links showed that each of the chains of type IX collagen, alpha 1(IX), alpha 2(IX), and alpha 3(IX), contained a site of cross-linking at the amino terminus of the COL2 triple-helix to which the alpha 1(II)N-telopeptide could bond. The alpha 3(IX)COL2 domain alone also had an attachment site for the alpha 1(II)C-telopeptide. The distance between the alpha 1(II)N-telopeptide and alpha 1(II)C-telopeptide interaction sites, 137 residues, is equal to the length of the hole zone (0.6D) in a type II collagen fibril. This implies an antiparallel type II to type IX cross-linking relationship. Peptide analysis also revealed an unknown amino acid sequence linked to the COL2 cross-linking domains in both the alpha 1(IX) and alpha 3(IX) chains. Using antibodies to this novel peptide, its origin in the collagen alpha 3(IX)NC1 domain was established. In summary, the results confirm extensive covalent cross-linking between type IX and type II collagen molecules and reveal the existence of type IX-type IX bonding. These data provide a molecular basis for the proposed function of type IX collagen as a critical contributor to the mechanical stability and resistance to swelling of the collagen type II fibril framework of cartilage.     

A heterozygous collagen defect in a variant of the Ehlers-Danlos syndrome type VII. Evidence for a deleted amino-telopeptide domain in the pro-alpha 2(I) chain

A structural defect in the alpha 2(I) chain of type I collagen was characterized in a new case of the Ehlers-Danlos syndrome type VII. The patient's skin, fascia, and bone collagens all showed an abnormal additional chain, pN-alpha 2(I)s, running slower than the alpha 2(I) chain on electrophoresis. The extension was shown to be on the amino-terminal fragment of pN-alpha (I)s by cleavage with human collagenase, but pepsin was unable to convert pN-alpha 2(I)s to alpha 2(I). Skin collagen was 4-fold more extractable and contained fewer beta-dimers and a lower concentration of cross-linking amino acids than control skin collagen. Electron micrographs of both dermis and bone showed markedly irregular ragged outlines of the collagen fibrils in cross-section, although the patient had no clinical signs of bone disease. Procollagen secreted by her skin fibroblasts in culture showed equal amounts of the normal and abnormal alpha 2(I) chains on pepsin digestion. Before pepsin, the pN-alpha 2(I) component ran as a doublet on electrophoresis; pepsin removed only the normal slower chain. The suspected deletion in pN-alpha 2(I)s was traced by CNBr peptide analysis to the N-propeptide fragment, which behaved on electrophoresis about 15-20 residues smaller than that from the normal pN-alpha 2(I) chain. The simplest genetic explanation is a spontaneous heterozygote in which one normal and one abnormal allele for the pro-alpha 2(I) gene are expressed, the protein defect being a deletion of the junction domain that spans the N-propeptidase cleavage site and the N-telopeptide cross-linking sequence.     

Characterization of the receptor for insulin-like growth factor II in bone cells

We have previously shown that insulin-like growth factor II (IGF-II) is produced by bone cells and that IGF-II stimulates cell proliferation and collagen synthesis in bone cells. We now extend these in vitro findings by demonstrating specific IGF-II binding to bone cells derived from newborn mouse calvaria and embryonic chick calvaria. The kinetics of [125I] IGF-II binding in embryonic chick calvaria cells showed time and temperature dependence. Scatchard analysis of [125I]IGF-II binding to chick calvaria cells showed an apparent Kd of 1.4 x 10(-10) M, with a calculated receptor site concentration of 40,000/cell. The specificity characteristics showed that IGF-II was significantly more potent than IGF-I or insulin in displacing IGF-II tracer. Competition for binding of [125I]IGF-II by unlabeled IGF-II showed a dose-dependent displacement between 0.5 and 25 ng/ml. Fifty percent displacement of [125I]IGF-II binding to chick and mouse calvarial cells was achieved at 1-2 ng/ml; 90% of specific binding of [125I]IGF-II was displaceable in the presence of 125 ng/ml of unlabeled IGF-II. IGF-I showed less than 5% cross reactivity for displacement of [125I]IGF-II binding to chick and mouse bone cells. Type II receptor inhibitory antibodies, R-II-PAB1 inhibited the binding of [125I]IGF-II to mouse bone cells and H-35 rat hepatoma cells (which contain type II but not type I receptors) in a dose-dependent manner. R-II-PAB1 also inhibited basal cell proliferation as well as IGF-II-, IGF-I-, and fibroblast growth factor (FGF)-induced cell proliferation in mouse bone cells. In chick calvaria bone cells and TE89 human osteosarcoma cells, R-II-PABI inhibited neither binding of [125I]IGF-II nor IGF-II-induced cell proliferation. These results together with our findings that IGF-II increased chick bone cell proliferation in the presence of maximal doses of IGF-I suggest that at least part of the mitogenic action of IGF-II is mediated through type II rather than type I receptors in bone cells.     

A base substitution at the splice acceptor site of intron 5 of the COL1A2 gene activates a cryptic splice site within exon 6 and generates abnormal type I procollagen in a patient with Ehlers-Danlos syndrome type VII.

The dermal type I collagen of a patient with Ehlers-Danlos type VIIB (EDS-VIIB) contained normal alpha 2(I) chains and mutant pN-alpha 2(I)' chains in which the amino-terminal propeptide (N-propeptide) remained attached to the alpha 2(I) chain. Similar alpha 2(I) chains were produced by cultured dermal fibroblasts. Amino acid sequencing of tryptic peptides, prepared from the mutant amino-terminal pN-alpha 2(I) CB1' peptide, indicated that five amino acids, including the N-proteinase (the specific proteinase that cleaves the procollagen N-propeptide) cleavage site, had been deleted from the junction of the N-propeptide and the N-telopeptide (the nonhelical domain at the amino-terminus of the alpha chains of fully processed type I polypeptide chains) of the mutant pro-alpha 2(I)' chain. The corresponding 15 nucleotides, which were deleted from approximately half of the alpha 2(I) cDNA polymerase chain reaction products, of the alpha 2(I) cDNA polymerase chain reaction products, were encoded by the +1 to +15 nucleotides of exon 6 of the normal alpha 2(I) gene (COL1A2). These 15 nucleotides were deleted in the splicing of alpha 2(I) pre-mRNA to mRNA as a result of inactivation of the 3' splice site of intron 5 by an AG to AC mutation and the activation of a cryptic AG splice acceptor site corresponding to positions +14 and +15 of exon 6. Loss of the N-proteinase cleavage site explained the persistence of the pN-alpha 2(I)' chains in the dermis and in fibroblast cultures. Collagen production by cultured dermal fibroblasts was doubled, possibly due to reduced feedback inhibition by the N-propeptides. In contrast to previously reported cases of EDS-VIIB, Lys5 of the N-telopeptide was not deleted and appeared to take part in the formation of intramolecular cross-linkages. However, increased collagen solubility and abnormal extraction profiles of the mutant type I collagen molecules indicated that collagen cross-linking was abnormal in the dermis. The proband and her son were heterozygous for the mutation. It is likely that the heterozygous loss of the N-proteinase cleavage site, with persistence of a shortened N-propeptide, was the major factor responsible for the EDS-VIIB phenotype.     

Synthesis of type III collagen by fibroblasts from the embryonic chick cornea

Synthesis of collagen types I, II, III, and IV in cells from the embryonic chick cornea was studied using specific antibodies and immunofluorescence. Synthesis of radioactively labeled collagen types I and III was followed by fluorographic detection of cyanogen bromide peptides on polyacrylamide slab gels and by carboxymethylcellulose chromatography followed by disc gel electrophoresis. Type III collagen had been detected previously by indirect immunofluorescence in the corneal epithelial cells at Hamburger-Hamilton stages 20--30 but not in the stroma at any age. Intact corneas from embryos older than stage 30 contain and synthesize type I collagen but no detectable type III collagen. However, whole stromata subjected to collagenase treatment and scraping (to remove epithelium and endothelium) and stromal fibroblasts from such corneas inoculated in vitro begin synthesis of type III collagen within a few hours while continuing to synthesize type I collagen. As demonstrated by double-antibody staining, most corneal fibroblasts contain collagen types I and III simultaneously. Collagen type III was identified biochemically in cell layers and media after chromatography on carboxymethylcellulose be detection of disulfide-linked alpha l (III)3 by SDS gel electrophoresis. The conditions under which the corneal fibroblasts gain the ability to synthesize type III collagen are the same as those under which they lose the ability to synthesize the specific proteoglycan of the cornea: the presence of corneal-type keratan sulfate.     

Selective adhesion of macrophages to denatured forms of type I collagen is mediated by scavenger receptors.

Macrophages (Mφs) are multifunctional immune cells which are involved in the regulation of immune and inflammatory responses, as well as in tissue repair and remodeling. In tissues, Mφs reside in areas which are rich in extracellular matrix (ECM), the structural component which also plays an essential role in regulating a variety of cellular functions. A major ECM protein encountered by Mφs is type I collagen, the most abundant of the fibril-forming collagens. In this study, the adhesion of RAW 264.7 murine Mphis to native fibrillar, monomeric, and denatured type I collagen was investigated. Using atomic force microscopy, structural differences between fibrillar and monomeric type I collagen were clearly resolved. When cultured on fibrillar type I collagen, Mphis adhered poorly. In contrast, they adhered significantly to monomeric, heat-denatured, or collagenase-modified type I collagen. Studies utilizing anti-beta1 and -beta2 integrin adhesion-blocking antibodies, RGD-containing peptides, or divalent cation-free conditions did not inhibit Mphi; adhesion to monomeric or denatured type I collagen. However, macrophage scavenger receptor (MSR) ligands and anti-MSR antibodies significantly blocked Mphi; adhesion to denatured and monomeric type I collagen strongly suggesting the involvement of the MSR as an adhesion molecule for denatured type I collagen. Further analysis by Western blot identified the MSR as the primary receptor for denatured type I collagen among Mphi; proteins purified from a heat-denatured type I collagen affinity column. These findings indicate that Mphis adhere selectively to denatured forms of type I collagen, but not the native fibrillar conformation, via their scavenger receptors.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo.

The aim of this work was to prepare specific antibodies against skin and bone collagen (type I) and cartilage collagen (type II) for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Antibodies against native type I collagen from chick cranial bone, and native pepsin-extracted type II collagen from chick sternal cartilage were raised in rabbits, rats, and guinea pigs. The antibodies, purified by cross-absorption on the heterologous collagen type, followed by absorption and elution from the homologous collagen type, were specific according to passive hemagglutination tests and indirect immunofluorescence staining of chick bone and cartilage tissues. Antibodies specific to type I collagen labeled bone trabeculae from tibia and perichondrium from sternal cartilage. Antibodies specific to type II collagen stained chondrocytes of sternal and epiphyseal cartilage, whereas fluorescence with intercellular cartilage collagen was obtained only after treatment with hyaluronidase. Applying type II collagen antibodies to sections of chick embryos, the earliest cartilage collagen found was in the notochord, at stage 15, followed by vertebral collagen secreted by sclerotome cells adjacent to the notochord from stage 25 onwards. Type I collagen was found in the dermatomal myotomal plate and presumptive dermis at stage 17, in limb mesenchyme at stage 24, and in the perichondrium of tibiae at stage 31.     

Cross-linking of collagen I by tissue transglutaminase provides a promising biomaterial for promoting bone healing

Transglutaminases (TGs) stabilize proteins by the formation of ε(γ-glutamyl)lysine cross-links. Here, we demonstrate that the cross-linking of collagen I (COL I) by tissue transglutaminase (TG2) causes an alteration in the morphology and rheological properties of the collagen fibers. Human osteoblasts (HOB) attach, spread, proliferate, differentiate and mineralize more rapidly on this cross-linked matrix compared to native collagen. When seeded on cross-linked COL I, HOB are more resistant to the loss of cell spreading by incubation with RGD containing peptides and with α1, α2 and β1 integrin blocking antibodies. Following adhesion on cross-linked collagen, HOB show increased phosphorylation of the focal adhesion kinase, and increased expression of β1 and β3 integrins. Addition of human bone morphogenetic protein to HOB seeded on TG2 cross-linked COL I enhanced the expression of the differentiation marker bone alkaline phosphatase when compared to cross-linked collagen alone. In summary, the use of TG2-modified COL I provides a promising new scaffold for promoting bone healing.     

A structural mutation of the collagen alpha 1(I)CB7 peptide in lethal perinatal osteogenesis imperfecta

Structurally abnormal type I collagen was identified in the dermis, bone, and cultured fibroblasts obtained from a baby with lethal perinatal osteogenesis imperfecta. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated that the alpha 1(I)CB7 peptide from the alpha 1(I)-chain of type I collagen existed in a normal form and a mutant form with a more basic charge distribution. This heterozygous peptide defect was not detected in the collagens from either parent. The defect was localized to a 224-residue region at the NH2 terminus of the alpha 1(I)CB7 peptide by mammalian collagenase digestion. Analysis of unhydroxylated collagens produced in cell culture indicated that the mutant alpha 1(I)CB7 migrated faster on electrophoresis suggesting that the abnormality may be a small deletion or a mutation that alters sodium dodecyl sulfate binding. The post-translational hydroxylation of lysine residues was increased in the CB7 peptide and also in peptides CB3 and CB8 which are toward the NH2 terminus of the alpha 1(I)-chain. The COOH-terminal CB6 peptide was normally hydroxylated. These findings support the proposal that the lysine overhydroxylation resulted from a perturbation of helix propagation from the COOH to NH2 terminus of the collagen trimer caused by the structural defect in alpha 1(I)CB7.     

Binding of soluble type I collagen to fibroblasts: specificities for native collagen types, triple helical structure, telopeptides, propeptides, and cyanogen bromide-derived peptides

Unlabeled collagenous proteins were quantified as inhibitors of binding of native, soluble, radioiodinated type I collagen to the fibroblast surface. Collagen types IV, V a minor cartilage isotype (1 alpha 2 alpha 3 alpha), and the collagenlike tail of acetylcholinesterase did not inhibit binding. Collagen types II and III behaved as competitive inhibitors of type I binding. Denaturation of native collagenous molecules exposed cryptic inhibitory determinants in the separated constituent alpha chains. Inhibition of binding by unlabeled type I collagen was not changed by enzymatic removal of the telopeptides. Inhibitory determinants were detected in cyanogen bromide-derived peptides from various regions of helical alpha 1 (I) and alpha 1(III) chains. The aminoterminal propeptide of chick pro alpha 1(I) was inhibitory for binding, whereas the carboxyterminal three-chain propeptide fragment of human type I procollagen was not. The data are discussed in terms of the proposal that binding to surface receptors initiates the assembly of periodic collagen fibrils in vivo.     

Collagen defects in lethal perinatal osteogenesis imperfecta.

Quantitative and qualitative abnormalities of collagen were observed in tissues and fibroblast cultures from 17 consecutive cases of lethal perinatal osteogenesis imperfecta (OI). The content of type I collagen was reduced in OI dermis and bone and the content of type III collagen was also reduced in the dermis. Normal bone contained 99.3% type I and 0.7% type V collagen whereas OI bone contained a lower proportion of type I, a greater proportion of type V and a significant amount of type III collagen. The type III and V collagens appeared to be structurally normal. In contrast, abnormal type I collagen chains, which migrated slowly on electrophoresis, were observed in all babies with OI. Cultured fibroblasts from five babies produced a mixture of normal and abnormal type I collagens; the abnormal collagen was not secreted in two cases and was slowly secreted in the others. Fibroblasts from 12 babies produced only abnormal type I collagens and they were also secreted slowly. The slower electrophoretic migration of the abnormal chains was due to enzymic overmodification of the lysine residues. The distribution of the cyanogen bromide peptides containing the overmodified residues was used to localize the underlying structural abnormalities to three regions of the type I procollagen chains. These regions included the carboxy-propeptide of the pro alpha 1(I)-chain, the helical alpha 1(I) CB7 peptide and the helical alpha 1(I) CB8 and CB3 peptides. In one baby a basic charge mutation was observed in the alpha 1(I) CB7 peptide and in another baby a basic charge mutation was observed in the alpha 1(I) CB8 peptide. The primary defects in lethal perinatal OI appear to reside in the type I collagen chains. Type III and V collagens did not appear to compensate for the deficiency of type I collagen in the tissues.