Glycine to tryptophan substitution in type I collagen in a patient with OI type III: a unique collagen mutation

We report a unique glycine substitution in type I collagen and highlight the clinical and biochemical consequences. The proband is a 9 year old Turkish boy with severely deforming osteogenesis imperfecta (OI). Biochemical analysis of (pro) collagen type I from a skin fibroblast culture showed both normal and overmodified α chains. Molecular analysis showed a G>T transversion in the COL1A2 gene, resulting in the substitution of glycine by tryptophan at position 277 of the α2(I) collagen chain. Glycine substitutions in type I collagen are the most frequent cause of the severe and lethal forms of OI. The phenotypic severity varies according to the nature and localisation of the mutation. Substitutions of glycine by tryptophan, which is the most voluminous amino acid, have not yet been identified in type I collagen or any other fibrillar collagen. The severe, though non-lethal OI phenotype associated with this mutation may appear surprising in view of the huge size of the tryptophan residue. The fact that the mutation resides within a so called "non-lethal" region of the α2(I) collagen chain supports a regional model in phenotypic severity for α2(I) collagen mutations, in which the phenotype is determined primarily by the nature of the collagen domain rather than the type of glycine substitution involved.


Keywords: osteogenesis imperfecta; COL1A2; tryptophan; collagen     

Chemical differences between individual human cold agglutinins

The μ- and κ-chains from four human cold agglutinins having anti-I specificity have been analysed:

The κ-chains have either Asp or Glu, but not both, at the N-terminus. This provides additional support for the view that cold agglutinins are of monoclonal origin.

Both heavy and light chains from cold agglutinins vary as much in amino acid composition as do monoclonal chains of a given type chosen at random. The chemical individuality of cold agglutinin μ-chains is shown also by differences in hexose, fucose and glucosamine content.

The apparent lack of correlation between overall antibody activity and chemical composition of constituent peptide chains indicates the need for close definition of combining specificity in structural studies of cold agglutinins.

     

Synthesis of immunoglobulins by biopsied tissues and cell lines from Burkitt's lymphoma

Cell suspensions of Burkitt''s lymphomas and cell lines derived from the same tumours were compared for immunoglobulin synthesis by analysis of the culture fluid. Thirty-one out of fifty tumour cell suspensions (i.e. twenty-one out of thirty-five patients) synthesized IgG (γ-chains) with type κ and/or type λ light chains; IgM synthesis was found in only five cases. Of the Burkitt''s lymphoma cell lines, twelve out of nineteen synthesized IgG (γ-chains); type κ light chains were produced by ten of these cell lines and type λ light chains by three. Only three cell lines synthesized μ chains. IgA synthesis was not detected in any of the biopsied tissues or cell lines.Comparison of the immunoglobulin synthesis by the cells of the biopsied tissue and the derived cell line showed very good agreement. This leads to the conclusion that the pattern of immunoglobulins synthesized by Burkitt''s lymphoma cell lines is representative of the original tumour.Investigation of cells from repeat biopsies, and serial testing of the derived cell lines showed that the capacity to synthesize particular immunoglobulins and chains remained constant.The fact that many of the biopsied tumour tissues and cell lines synthesized more than one immunoglobulin, or different classes of heavy chains and types of light chains, raises the question whether these immunoglobulin-producing cells originate from one or more cells.     

Total absence of the alpha2(I) chain of collagen type I causes a rare form of Ehlers-Danlos syndrome with hypermobility and propensity to cardiac valvular problems

Background

Heterozygous mutations in the COL1A1 or COL1A2 gene encoding the α1 and α2 chain of type I collagen generally cause either osteogenesis imperfecta or the arthrochalasis form of Ehlers‐Danlos syndrome (EDS). Homozygous or compound heterozygous COL1A2 mutations resulting in complete deficiency of the proα2(I) collagen chains are extremely rare and have been reported in only a few patients, albeit with variable phenotypic outcome.

Methods

The clinical features of the proband, a 6 year old boy, were recorded. Analysis of proα and α‐collagen chains was performed by SDS‐polyacrylamide gel electrophoresis using the Laemmli buffer system. Single stranded conformation polymorphism analysis of the proband''s DNA was also carried out.

Results

In this report we show that complete lack of proα2(I) collagen chains can present as a phenotype reminiscent of mild hypermobility EDS during childhood.

Conclusions

Biochemical analysis of collagens extracted from skin fibroblasts is a powerful tool to detect the subset of patients with complete absence of proα2(I) collagen chains, and in these patients, careful cardiac follow up with ultrasonography is highly recommended because of the risk for cardiac valvular problems in adulthood.     

Characterization of conformation independent antigenic determinants in the triple-helical part of calf and rat collagen

About 20 per cent of the antibodies in rabbit antisera to native calf or rat collagen exhibited affinity for denatured rabbit collagen and could be isolated by immunoadsorption. Such antibodies reacted with the unfolded α1-chain as well as with the α2-chain of collagen. Inhibition experiments suggested that the two kinds of polypeptide chains are not completely equivalent in their antigenic determinants. These determinants were not significantly influenced by a treatment of native collagen with pronase, a procedure known to remove short, non-helical sequences at both ends of the molecule. The results suggested that the antigenic determinants are conformation independent. They are, however, mainly located in the middle region of collagen, having a rather complex conformational structure.

Cyanogen bromide cleavage of collagen did not impair the serologic activity of these determinants but with one exception none of the individual cyanogen bromide peptides possessed the full activity of the entire α-chain. However, most of the peptides could be agglutinated by the antibodies when put onto tanned red cells. Inhibition studies of these agglutination reactions clearly demonstrated that virtually all of the peptides carry unique antigenic determinants, which occasionally are shared by a few other peptides. Additional evidence for heterogeneity was obtained by further cleavage of the cyanogen bromide peptides with proteases. The minimum number of antigenic determinants thus estimated in calf collagen was nine. Evidence is provided that their structure in most cases does not correspond to sequences of the type Gly-Pro-X.

     

The behavior of (14C)-D-penicillamine in collagen metabolism.

The most important effect of penicillamine on collagen metabolism is the reduction of collagen crosslinking. However, even after long time application of penicillamine, collagen is crosslinked to a certain degree. After intravenous injection of a trace dose of (14C) labelled D-penicillamine it can be determined that this substance is rapidly bound to neutral salt soluble, acetic acid soluble and urea soluble collagen fractions and to a lesser extent to insoluble collagen as well. The amount of penicillamine which binds to any of the collagen fractions depends on the turnover rate. When different tissues are compared, penicillamine seems to have the greatest affinity to tissues with a high collagen turnover. Further studies of neutral salt soluble collagen by CM-cellulose chromatography revealed a stable linkage of penicillamine to collagen alpha chains.     

The antigenicity of native and tyrosylated neutral-salt-soluble rat collagen

Neutral-salt-soluble collagen was extracted from rat skin and purified by repeated precipitation, resolution and dialysis. Part was reacted with N-carboxytyrosine anhydride and a collagen derivative containing 2·6 per cent tyrosine was recovered. Enrichment with tyrosine did not alter the optical rotation, denaturation temperature or electrophoretic mobility of the collagen.

The antigenic properties of native and tyrosylated rat collagen were studied in rabbits and guinea-pigs by micro-complement fixation, tanned cell agglutination and agglutination-inhibition, passive cutaneous anaphylaxis and immediate and delayed skin hypersensitivity. The antigenicity of native collagen was demonstrated. Enrichment with a limited amount of tyrosine enhanced its antigenicity without altering its antigenic specificity and permitted a detailed analysis of the overall specificity of the immunological reaction. Use of the products of controlled degradation of collagen in the immunoassay systems implicitly defined the collagen molecule as responsible for the immune reaction. Collagenase-digestion products still possessed antigenic capacity.

     

The structural basis of cell-mediated immunological reactions of collagen: Characteristics of cutaneous delayed hypersensitivity reactions in specifically sensitized guinea-pigs

Cell-mediated immunological properties of collagen were studied in guinea-pigs employing cutaneous delayed hypersensitivity reactions. The animals were sensitized by a single injection of highly purified native collagen in Freund's complete adjuvant (FCA). Reactivity could be induced with 150 μg of calf collagen. Maximal reactivity was obtained 20 days after sensitization and persisted for more than 3 months. Histologically, the reactions displayed the typical features of delayed reactions with infiltration of predominantly mononuclear cells. No reactivity was induced in animals injected with FCA alone, with collagen in Freund's incomplete adjuvant (FIA), or with guinea-pig collagen in Freund's complete adjuvant. Reactivity was impaired when carrageenan was injected intraperitoneally at the time of challenge. Cyanogen bromide digested collagen was still reactive in sensitization as well as in elicitation. The reaction was found to be species specific in the sense that maximal reactions were obtained when the challenging collagen was from the same species as the sensitizing preparation. In vitro, denatured rat collagen was found to inhibit the migration of macrophages from specifically sensitized animals. By alterations of the immunization schedule antibodies, reactive with collagen in a haemagglutination system, could be induced.

The system lends itself to a comparative investigation of the structural requirements on a natural protein for the induction of the cell-mediated and the humoral immune response.

     

Involvement of more than a single polypeptide chain in the helical antigenic determinants of collagen

Rat antibodies to native triple helical calf collagen were used to study the structural requirements of helical antigenic determinants revealed in this particular model. Renaturation of random-coiled chains of collagen in the original proportion of two α1-chains and one α2-chain to triple-helical structures is accompanied by complete recovery of antigenic activity. Although physically similar to native collagen, triple-helical structures prepared from individual chains alone were inactive or exhibited a strongly reduced activity. These results were interpreted as to the involvement of parts of the α1 and α2-chains in several dinstinct helical antigenic determinants of collagen. Further studies with collagenase-derived, triple helical fragments of variable length revealed successive loss of antigenic activity upon reduction in size. However, the smallest fragment, having a length of 78 nm, still contained some of the original antigenic determinants. The antibodies to helical antigenic determinants also showed rather limited interspecies cross-reactions as opposed to the very strong cross-reactions found previously between antigenic determinations of sequential nature in random-coiled α-chains. Helix formation of two different types of α-chains essentially masks these cross-reacting structures.     

Specific suppression of delayed hypersensitivity skin reactions to collagen in guinea-pigs after immunization with collagen and Freund''s incomplete adjuvant.

Partial suppression of cutaneous delayed hypersensitivity reactions to collagen in guinea-pigs was induced by pre-immunization with collagen and FIA. This suppression is specific since: (a) pretreatment with OA and FIA or FIA alone did not cause suppression of skin reactions to collagen; (b) suppression was observed only if the collagen used for pretreatment was from the same species as that employed for sensitization for delayed hypersensitivity reactions; and (c) animals with depressed skin reactivity to collagen reacted normally to PPD. The suppression is not mediated by inducible, circulating antibodies to collagen since: (a) antibody titres measured by passive haemagglutination did not correlate with the degree of suppression; (b) suppression was observed with collagen in random coil conformation which sensitizes guinea-pigs for delayed hypersensitivity skin reaction but does not induce antibodies to denatured collagen; (c) best suppression was obtained if the animals were pretreated with collagen and FIA 3 days before the sensitizing injection; and (d) passively transferred antibody from animals with suppressed skin reactivity did not suppress skin reactivity of animals made hypersensitive to collagen by injection of collagen and FCA.     

Immunological studies of an atypical (myeloma) immunoglobulin

An 8S myeloma component, isolated from serum of a patient with myelomatosis is described, which appears to have no antigenic determinants in common with human, α-, δ-, γ- or μ-polypeptide chains as revealed by immuno-electrophoresis and Ouchterlony gel diffusion analysis.

The myeloma protein migrates in the fast γ-region on electrophoresis at pH 8.6 and has an elution volume on Sephadex G-200 similar to that of 6.5S IgA.

The isolated myeloma component has an approximate molecular weight of 200,000 and a total carbohydrate content of 10.7 per cent.

Reduction with β-mercaptoethanol and acid dissociation yields light polypeptide chains of Type L and a carbohydrate-rich component, in the ratio of 1:4.

Antisera specific to determinants on the heavy chains of the myeloma protein showed no reaction with the immunoglobulins A, D, G or M. Instead unique determinants were found on the heavy polypeptide chains.

     

Epitope mapping of anti-glomerular basement membrane (GBM) antibodies with synthetic peptides

Autoantibodies to the non-collagenous (NC1) domain of the α3(IV)-chain of type IV collagen are found in sera from patients with anti-GBM nephritis. These antibodies have been shown to be pathogenic. In this study the antibody specificity has been investigated in patients with Goodpasture’s syndrome and from a patient with atypical anti-GBM antibodies, recognizing the α1(IV)-chain only. Overlapping synthetic peptides, covering the complete NC1 domains of the α1(IV)- and α3(IV)-chains were used in sandwich ELISA and competitive ELISA. None of the Goodpasture sera showed reactivity to the synthetic peptides. However, antibodies from the patient with atypical anti-GBM antibodies recognized a 20 amino acid peptide from the α1(IV)-chain. The reactive peptide was further narrowed down with glycine substitution of the different amino acids. We have localized the epitope to the four last C-terminal amino acids of the α1(IV)-chain, with the sequence 1754-MRRT. The two arginine residues were found to be essential for antibody binding. Threonine is important, while methionine is of less importance. These four amino acids are also determined to be the smallest peptide that could inhibit the binding of the autoantibodies to the native α1(IV)-chain. This study shows that overlapping peptides can be used to map linear epitopes. However, for conformational epitopes such as the Goodpasture epitope, other methods must be used. It would be prognostically important to know the fine specificity of anti-GBM antibodies, since the patient with anti-α1(IV) antibodies had a mild disease, while the Goodpasture patients with anti-α3(IV) antibodies had a rapidly progressive disease.     

Calcium alginate enhances wound healing by up-regulating the ratio of collagen types I/III in diabetic rats

Calcium alginate has been proved to favor the skin ulcer healing and collagen synthesis was a critical factor for the wound closure. The present study was to elucidate the mechanism of calcium alginate on the diabetes skin ulceration. Calcium alginate dressing was applied daily on the full-thickness exercising wound created on the back of diabetic rat model as Alg-group (n=6), and the vaseline dressing was used as control (n=6). Rats were respectively sacrificed and the wound tissues were removed and used for the evaluation of various biochemical analysis contained collagen (type I and III) by Western blotting and hydroxyproline level changes by ELISA assay at 3 d, 7 d and 14 d after wounding. The expression of skin collagen I in Alg-group was enhanced from day 3 (0.66±0.25 vs. 0.42±0.09, P<0.05) to day 14 (1.09±0.14 vs. 0.78±0.16, P<0.05). However, no significant difference of collagen III expression was found between two groups during wound healing (P>0.05). And the ratio of collagen I/III in Alg-group was greater than that of Vas-group at day 7 (1.07±0.31 vs. 0.77±0.11, P<0.05) and 14 (1.18±0.30 vs. 0.83±0.14, P<0.05). The hydroxyproline level in skin homogenate of Alg-group was higher than that of Vas-group from day 3 (30.29±0.92 ng/ml vs. 27.52±0.83 ng/ml, P<0.05) to day 14 (89.58±4.97 ng/ml vs. 79.30±4.42 ng/ml, P<0.05). Calcium alginate accelerates the process of wound healing through improving type I collagen synthesis and increasing ratio of collagen I/III in diabetic rats.     

Kappa and lambda receptor sites on single lymphocytes

Light chain receptors on human circulating lymphocytes of thirty-three normal individuals were visualized by the immunocyto-adhesion reaction in which human (anuclear) erythrocytes coated with κ chains and chicken (nucleated) erythrocytes coated with λ chains (or vice versa) were used. 4·4 (±2·3)% of the lymphocytes had κ and 4·15 (±2·4)% had λ receptors, when the lymphocytes were sensitized with antisera to either κ and λ chains. When both antisera were used simultaneously only 5·3 (±3)% were found to have receptors and the majority of cells carried both κ and λ receptors. These findings suggest that in these cells the genes for the constant regions of both light chains are active.     

Enterococcus faecalis adhesin, ace, mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen type I

Adhesin-mediated binding to extracellular matrix (ECM) proteins is thought to be a crucial step in the pathogenic process of many bacterial infections. We have previously reported conditional adherence of most Enterococcus faecalis isolates, after growth at 46°C, to ECM proteins collagen types I and IV and laminin; identified an E. faecalis-specific gene, ace, whose encoded protein has characteristics of a bacterial adhesin; and implicated Ace in binding to collagen type I. In this study, we constructed an ace disruption mutant from E. faecalis strain OG1RF that showed marked reduction in adherence to collagen types I and IV and laminin when compared to the parental OG1RF strain after growth at 46°C. Polyclonal immune serum raised against the OG1RF-derived recombinant Ace A domain reacted with a single ~105-kDa band of mutanolysin extracts from OG1RF grown at 46°C, while no band was detected in extracts from OG1RF grown at 37°C, nor from the OG1RF ace mutant grown at 37 or 46°C. IgGs purified from the anti-Ace A immune serum inhibited adherence of 46°C-grown E. faecalis OG1RF to immobilized collagen type IV and laminin as well as collagen type I, at a concentration as low as 1 μg/ml, and also inhibited the 46°C-evoked adherence of two clinical isolates tested. We also showed in vitro interaction of collagen type IV with Ace from OG1RF mutanolysin extracts on a far-Western blot. Binding of recombinant Ace A to immobilized collagen types I and IV and laminin was demonstrated in an enzyme-linked immunosorbent assay and was shown to be concentration dependent. These results indicate that Ace A mediates the conditional binding of E. faecalis OG1RF to collagen type IV and laminin in addition to collagen type I.     

Anaphylactic reactions to univalent haptens

The question whether a univalent antigen may suffice for the elicitation of anaphylactic reactions is the subject of this study. We previously reported that certain univalent alpha substituted ε-DNP-lysine compounds can elicit such reactions. Evidence on this question has been extended to include the use of a number of aliphatic as well as a cyclic α substituent in ε-DNP-lysine.

The haptens most active in eliciting PCA responses were the 8 and 10 C α-acyl derivatives. A cyclic acyl compound (α-hexahydrobenzoyl-ε-DNP-lysine) was inactive.

A hypothesis is advanced to account for the release of histamine from the cells of sensitive animals by simple (1:1) complexing of antigen with antibody, based upon the structures and activities of the compounds tested.

     

Tissue transglutaminase contributes to interstitial renal fibrosis by favoring accumulation of fibrillar collagen through TGF-beta activation and cell infiltration

Renal fibrosis is defined by the exaggerated accumulation of extracellular matrix proteins. Tissue transglutaminase (TG2) modifies the stability of extracellular matrix proteins and renders the extracellular matrix resistant to degradation. In addition, TG2 also activates transforming growth factor-β (TGF-β). We investigated the involvement of TG2 in the development of renal fibrosis using mice with a knockout of the TG2 gene (KO). These mice were studied at baseline and 12 days after unilateral ureteral obstruction, which induced a significant increase in interstitial TG2 expression in wild-type mice (P < 0.001). Interstitial fibrosis was evident in both groups, but total and fibrillar collagen was considerably lower in KO mice as compared with wild-type (P < 0.001). Similarly, mRNA and protein expression of collagen I were significantly lower in KO animals (P < 0.05). A statistically significant reduction in renal inflammation and fewer myofibroblasts were observed in KO mice (P < 0.01). Free active TGF-β was decreased in KO mice (P < 0.05), although total (active + latent) TFG-β concentration did not differ between groups. These results show that mice deficient in TG2 are protected against the development of fibrotic lesions in obstructive nephropathy. This protection results from reduced macrophage and myofibroblast infiltration, as well as from a decreased rate of collagen I synthesis because of decreased TGF-β activation. Our results suggest that inhibition of TG2 may provide a new and important therapeutic target against the progression of renal fibrosis.     

Regulation of the atheroma-enriched protein, SPRR3, in vascular smooth muscle cells through cyclic strain is dependent on integrin alpha1beta1/collagen interaction

Atherosclerotic plaques express high levels of small proline-rich repeat protein (SPRR3), a previously characterized component of the cornified cell envelope of stratified epithelia, where it is believed to play a role in cellular adaptation to biomechanical stress. We investigated the physiological signals and underlying mechanism(s) that regulate atheroma-enriched SPRR3 expression in vascular smooth muscle cells (VSMCs). We showed that SPRR3 is expressed by VSMCs in both human and mouse atheromas. In cultured arterial VSMCs, mechanical cyclic strain, but neither shear stress nor lipid loading induced SPRR3 expression. Furthermore, this upregulation of SPRR3 expression was dependent on VSMC adherence to type I collagen. To link the mechanoregulation of SPRR3 to specific collagen/integrin interactions, we used blocking antibodies against either integrin α1 or α2 subunits and VSMCs from mice that lack specific collagen receptors. Our results showed a dependence on the α1β1 integrin for SPRR3 expression induced by cyclic strain. Furthermore, we showed that integrin α1 but not α2 subunits were expressed on VSMCs within mouse lesions but not in normal arteries. Therefore, we identified the enrichment of the mechanical strain-regulated protein SPRR3 in VSMCs of both human and mouse atherosclerotic lesions whose expression is dependent on the collagen-binding integrin α1β1 on VSMCs. These data suggest that SPRR3 may play a role in VSMC adaptation to local biomechanical stress within the plaque microenvironment.     

The Behavior of (14C)-D-Penicillamine in Collagen Metabolism

The most important effect of penicillamine on collagen metabolism is the reduction of collagen crosslinking. However, even after long time application of penicillamine, collagen is crosslinked to a certain degree. After intravenous injection of a trace dose of (¹⁴C) labelled D-penicillamine it can be determined that this substance is rapidly bound to neutral salt soluble, acetic acid soluble and urea soluble collagen fractions and to a lesser extent to insoluble collagen as well. The amount of penicillamine which binds to any of the collagen fractions depends on the turnover rate. When different tissues are compared, penicillamine seems to have the greatest affinity to tissues with a high collagen turnover. Further studies of neutral salt soluble collagen by CM-cellulose chroma-tography revealed a stable linkage of penicillamine to collagen alpha chains.