A recurrent valpha17/vbeta10 TCR-expressing T cell clone is involved in the pathogenicity of collagen-induced arthritis in DBA/1 mice.

Collagen-induced arthritis (CIA) is an experimental model that mimics clinical and histological features of rheumatoid arthritis. In this disease, a crucial role in initiating the pathological changes has been assigned to T lymphocytes expressing the Th1 phenotype. Aiming at identifying type II collagen (CII)-specific T cells involved in CIA, T cell clones were generated in vitro from the lymph nodes (LN) of CII-immunized DBA / 1 mice. In three independent experiments, we repeatedly isolated CD4(+) Th1 clones recognizing the immunodominant epitope in the CB11 fragment of bovine CII and expressing a unique alpha betaTCR produced by the rearrangement of Valpha17/Jalpha20 and Vbeta10/Dbeta1.1/Jbeta2.5 gene segments. By reverse transcriptase-PCR, we demonstrated the presence of mRNA transcripts specific for the beta complementary-determining region 3 of this clonotype in the LN of the majority (73%) of mice with CIA whereas it was never detected in control animals. When transferred to CII-immunized DBA/1 mice, this recurrent Th1 clone augmented the incidence, aggravated significantly the clinical signs of CIA and greatly enhanced the anti-CII antibody response. Altogether, these results provide evidence that a CD4(+) Th1 clone belonging to the public arm of the response toward the immunodominant epitope of CII is involved in the cascade of events leading to CIA.

Characterization of selachian egg case collagen.

The egg case of the dogfish Scyliorhinus canicula is a remarkable collagenous structure that combines mechanical strength and toughness with high permeability to small molecules and ions. The collagenous lamellae that form over 80% of the thickness of the case wall are secreted by the D-zone of the nidamental (oviducal gland). An acid-soluble collagen extracted from this zone and partially purified ran as a single band on a native gel at pH 4.3. A single band of identical mobility was extracted from egg cases removed from the oviducal gland. SDS-PAGE of both extracts revealed a major component with an apparent molecular weight of 35 kDa and a minor component at 34 kDa. Neither of these components appeared to be glycosylated. Amino acid analysis of the partially purified collagen extracted from the oviducal gland revealed a composition similar to that of the collagenous lamellae of the egg case with glycine accounting for 16% and imino acids for 10% of the total residues. Partial N-terminal and internal sequences were obtained by Edman degradation for peptides extracted from the D-zone of the nidamental gland. Four of the internal sequence fragments showed repeated G-X-Y triplets showing them to be collagenous. These four fragments were novel but showed similarity to the triple-helical domains of mammalian type IV, X, and VI collagens. The noncollagenous N-terminal and pepsin-resistant sequences were unique, showing no significant similarity to known proteins in the database. Several possible N-myristoylation and phosphorylation sites were identified in the noncollagenous sequences.

Structural requirements for fibromodulin binding to collagen and the control of type I collagen fibrillogenesis--critical roles for disulphide bonding and the C-terminal region.

Fibromodulin belongs to the family of small, leucine-rich proteoglycans which have been reported to interact with collagens and to inhibit type I collagen fibrillogenesis. Decorin and fibromodulin exhibit a noticeable degree of sequence similarity. However, as previously reported [Font, B., Eichenberger, D., Rosenberg, L. M. & van der Rest, M. (1996) Matrix Biol. 15, 341-348] the domains of these molecules implicated in the interactions with type XII and type XIV collagens are different, these being the dermatan sulphate/chondroitin sulphate chain for decorin and the core protein for fibromodulin. At the present time the fibromodulin domains implicated in the interactions with fibrillar collagens remain unknown. In experiments reported here, we have sought to identify the structural requirements for fibromodulin interaction with collagen and for the control of type I collagen fibrillogenesis. Circular dichroism spectra and fibrillogenesis inhibition studies show that fibromodulin structure and its collagen fibrillogenesis control function are strictly dependent on the presence of intact disulphide bridge(s). In addition, we show that the binding of fibromodulin (or fibromodulin-derived fragments) to type I collagen is not necessarily correlated with fibrillogenesis inhibition. To isolate fibromodulin domains, the native proteoglycan was submitted to mild proteolysis. We have isolated an alpha-chymotrypsin-resistant fragment which contains the bulk of the N-terminal and central region of the molecule including the leucine-rich repeats 4 and 6 reported for decorin to be involved in type I collagen binding. This fragment does not bind to type I collagen. Using enzymes with different specificities, a number of large fragments of fibromodulin were obtained, suggesting a compact structure for this molecule which is relatively resistant to proteolysis. None of these N-glycosylated fragments were able to bind to type I collagen in co-sedimentation experiments. Taken together these results suggest that fibromodulin-type I collagen interactions leading to fibrillogenesis inhibition require more than one binding domain. One of these domains could be the C-terminal end of the molecule containing the disulphide loop which is absent in the chymotrypsin-resistant fragment.

Processing of type XI collagen. Determination of the matrix forms of the alpha1(XI) chain.

Type XI collagen is mainly found as a minor constituent in type II-containing fibrils and presents a alpha1(XI)alpha2(XI)alpha3(XI) stoichiometry. This molecule was shown to be partially processed in its intact tissue form. Moreover, alternative splicing has been demonstrated in the variable region of the N-terminal domain of alpha1(XI) and alpha2(XI) chains. In this work, the processing of a major intact form of alpha1(XI) from matrix laid down by chick chondrocytes in culture was identified using N-terminal sequencing and antibodies to synthetic peptides corresponding to the N-terminal propeptide cDNA-derived sequence. The results show that the fully processed form of alpha1(XI) begins at Gln254 of the N-terminal propeptide, seven residues before the end of the proline/arginine-rich protein region encoded by exon I (Zhidkova, N. I., Justice, S. K., and Mayne, R. (1995) J. Biol. Chem. 270, 9486-9493). This sequence is immediately followed by a sequence encoded by exon III. The processing takes place at an Ala-Gln sequence that corresponds to a consensus sequence for procollagen N-proteinase. The antibody raised against a sequence located within the region corresponding to exon IV (anti-P8) fails to recognize this fully processed form of the alpha1(XI) chain. It recognizes, however, two minor bands of high molecular mass. These results suggest that a major cartilage form of alpha1(XI) is the product of alternative splicing in which sequences encoded by both exons II and IV are skipped. The presence of a highly acidic subdomain encoded by exon III at the N terminus of the major form of the alpha1(XI) chain, as predicted by these data, provides potential sites for interaction of collagen XI with other molecules.

Flexilin: a new extracellular matrix glycoprotein localized on collagen fibrils.

We have immunopurified and characterized a new glycoprotein of the extracellular matrix, using a monoclonal antibody obtained after immunization with fibril-associated collagens extracted from bovine tendon. In polyacrylamide gels, the protein migrates at about 350 kDa molecular mass. The protein is insensitive to bacterial collagenase, and no disulfide-linked aggregates could be detected; sugars were stained with periodic acid-Schiff's reagent. Amino acid analysis and sequencing of tryptic peptides failed to detect any similarity with known proteins. By rotary shadowing experiments, the protein was observed as flexible, unbranched structures, approximately 150 nm long, with a small globule at one end. Investigation of the tissue distribution of the protein in fetal bovine tissues by immunofluorescence resulted in labeling in extracellular matrices with loosely packed collagen fibrils, such as the peritendineum, embryonic skin and kidney glomeruli; cornea, cartilage matrix and bone were not labeled. Ultrastructural immunolocalization in dermis and in mesangium of glomeruli showed that the protein always occurred in the vicinity of collagen fibrils. In view of its tissue distribution and molecular shape, we postulate that this protein is important in the properties of the extrafibrillar environment. By reference to its shape as observed by rotary shadowing, we propose the name 'flexilin' for this extracellular matrix glycoprotein.

Immunohistochemical and biochemical analyses of 20,000-25,000-year-old fossil cartilage.

A cracked, irregular pellicle adhering to fossilized bone excavated from the Enlène cave (Ariège) and estimated to date from 20,000-25,000 years BP was examined to verify its cartilaginous nature, suggested previously on the basis of optical and electron microscopic investigations. Immunolabeling of the organic component revealed the presence of type II and IX collagens, associated with residual glycosaminoglycans, in the external zone of the pellicle. The cartilaginous nature of the pellicle was also demonstrated by biochemical identification of type II collagen as the major protein in the demineralized sample: the amino acid compositions of insoluble and soluble fractions were similar to that of pure type II collagen; cyanogen-bromide-generated peptides, prepared after reduction of the sample, had an electrophoretic pattern similar to that of cyanogen bromide peptides derived from type II collagen. The amino acid sequences of four tryptic peptides were identical to the corresponding human type II sequences. It was impossible to isolate intact alpha chains. All of the solubilized fractions were composed of a wide range of low-molecular-mass peptides demonstrating significant degradation of the collagen molecules that was not reflected in the well-preserved fibrillar structure observed at the ultrastructural level. The mineral fraction, characterized by X-ray diffraction, consisted of apatite (as in sub-chondral bone) associated with contaminating poorly crystallized components originating from the cave sediment. Energy dispersive spectrometry showed that the cartilaginous zone contained three times less phosphorus and calcium than the underlying bone. These results confirm the cartilaginous nature of the sample and the preservation of tissue-specific components, and suggest that the process of fossilization is closely related to a mechanism of phosphatization.

Two-dimensional electrophoresis of intracellular and secreted protein synthesized by fetal bovine chondrocytes in high-density culture.

In order to study the mechanisms involved in the differentiation/dedifferentiation of chondrocytes, fetal bovine chondrocytes in high-density cultures were treated with retinoic acid, an agent known to modify the chondrocyte phenotype (10 mumol/L between day 2 to day 5 of culture). The synthesis of intracellular and secreted proteins was studied by two-dimensional electrophoresis in cell lysates and culture media after labeling with [35S]methionine for the last 14 h of culture. The proteins expressed in control and retinoic acid-treated cells were identified by microsequencing after "in-gel" tryptic digestion of the spot or by immunodetection with specific antibodies after two-dimensional gel blotting. Intracellular protein modifications included one of 56.9 kDa and with an isoelectric point (pI) of 5.8 whose synthesis was previously reported to be up-regulated by 75%. Microsequencing of two internal peptides did not reveal a known protein. Changes to the chondrocyte phenotype were also recorded in the culture medium, as a decrease in type II collagen synthesis and expression of the small proteoglycan, decorin. Several new spots were also observed after treatment with retinoic acid, including a large, diffuse spot, not yet characterized, with a mean molecular mass of 39 kDa and a pI of 4.5-5.0. Under our experimental conditions, retinoic acid induces morphological changes of the chondrocytes and dramatic changes in the synthesis of several intracellular and secreted proteins that predate the synthesis of collagen type I (the classical marker of chondrocyte dedifferentiation).

Onco-fetal/laminin-binding collagen from colon carcinoma: detection of new sequences.

We have recently identified an oncofetal-laminin binding collagen (OF/LB) composed of three alpha chains, with the apparent molecular mass of about 100 kDa each, but bearing different pI. One of the chains appears markedly acidic in a bidimensional electrophoretic system, where the NEPHGE is used as first dimension separating gel, while the two more basic chains have similar migration as alpha 1(III) and alpha 1(I) collagen chains, respectively. Sequence analyses have been performed on CNBr-peptides, derived from pepsinized triple helical molecules and on tryptic fragments obtained after in gel digestion of the acidic band. The research of sequence homology with computerized databases indicated that the acidic chain represents a gene product distinct from either type I, type III and other known collagen chains, while the identity of the other two chains remains to be fully determined.

Effect of retinoic acid on protein synthesis by foetal bovine chondrocytes in high-density culture: down-regulation of the glucose-regulated protein, GRP-78, and type II collagen.

The effect of 0.1-10 microM retinoic acid (RA) on foetal bovine chondrocytes was investigated in high-density cultures (0.6 x 10(6) cells/cm2). After 5 days of culture in ascorbate-free medium, control chondrocytes presented a typical rounded shape and synthesized type II, IX, XI and III collagens. After RA treatment on days 2-5 of culture, the cells exhibited a fibroblast-like shape and decreased synthesis of total protein (48%) and pepsinresistant proteins (60%) as determined by [35S]methionine labelling. Addition of RA was not followed by the expression of type I collagen, but induced quantitative changes in the synthesis of cartilage-specific collagens (II, IX and XI) as measured by direct autoradiography of the corresponding bands after SDS/PAGE. The main change was in type II collagen synthesis, with a 80% decrease in the cell-layer fraction and a 89% decrease in culture-medium fraction; inhibition of type IX and XI collagen synthesis was limited to 25 and 31% respectively. Modifications to intracellular proteins induced by RA were determined by using two-dimensional electrophoresis associated with a computerized imaging system. Synthesis of one of the more abundant proteins (pI 4.8; 78 kDa) was decreased by 75% after RA treatment. This protein was characterized by micro-sequencing as the glucose-regulated protein 78 (GRP 78). It was reported previously to bind denatured collagen and mutated type I procollagen molecule and to function as a molecular chaperone for collagen molecules. It remains to demonstrate whether the parallel down-regulation of GRP 78 and type II collagen observed here corresponds to a co-ordinate regulation of these two proteins.

Diversity in the processing events at the N-terminus of type-V collagen.

The processing of human collagen type-V chains was studied using anti-peptide polyclonal antibodies raised against peptide sequences at the N-terminal non-triple-helical region of pro-alpha 1(V) and pro-alpha 2(V) chains. The anti-peptide polyclonal antibody raised against positions 48-57 of the N-terminal alpha 2(V) sequence recognized the mature form of the human alpha 2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-alpha 2(V) and pN-alpha 2(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-alpha 2(V) collagen chain from this cell line migrated during electrophoresis with the alpha 2(V) chain obtained from tissues. This demonstrates that the alpha 2(V) chain in tissues is incompletely processed and is present as the pN-alpha 2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclonal antibody raised against residues at positions 284-299 of the N-terminal alpha 1(V) human sequence failed to recognize the mature form of the alpha 1(V) chain while it reacted with the pN-alpha 1(V) collagen chain form. These results suggest that the alpha 1(V) chain undergoes a processing event in the N-terminal region that involves the removal of at least the first 284 residues. Amino acid sequence analysis was performed on cyanogen-bromide-generated or trypsin-generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower-migrating band corresponding to the intact alpha 1(V) chain gave, as expected, only sequences corresponding to the alpha 1(V) chain. However, the band previously considered to be the intact alpha 2(V) chain also gave sequences for the alpha 1(V) chain in addition to the alpha 2(V) chain. This result indicates the presence in tissue extracts of a further processed form of alpha 1(V) chain which migrates with the intact alpha 2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1:1 ratio whereas, after the pepsin digestion to remove non-collagenous regions, two bands were observed with an alpha 1(V)/alpha 2(V) chain ratio of 3:1. These results indicate that the alpha 1(V) chain exists in an additional stoichiometry, different from [alpha 1(V)]2 alpha 2(V).(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of type I procollagen N-proteinase from fetal bovine tendon and skin. Purification of the 500-kilodalton form of the enzyme from bovine tendon.

Procollagen N-proteinase (EC 3.4.24.14) is the enzyme that specifically cleaves the NH2-terminal propeptides from type I procollagen. Two forms of N-proteinase with apparent molecular sizes of 300 and 500 kDa were found in partially purified preparations from fetal bovine tendon extracts. The 500-kDa form of enzyme was purified 16,000-fold with a recovery of 8% from the extracts of the tendons by six purification steps. The purified enzyme was a neutral, Ca(2+)-dependent proteinase (5-10 mM) that was inhibited by metal chelators. The 500-kDa enzyme contained unreduced polypeptides of 58, 125, 170, and 190 kDa which were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Electron microscopic study indicated that the enzyme molecules were generally globular and had diameters of 33 +/- 4 nm. Other properties of the 500-kDa enzyme were: 1) the Km for type I procollagen is 35 nM at pH 7.5 and 35 degrees C, and the kcat is 290 h-1; 2) the activation energy for reaction with type I procollagen is 10,050 cal mol-1; 3) the isoelectric point is 3.8; 4) the enzyme cleaves the NH2-terminal propeptides of type II procollagen as well as type I procollagen but not of type III procollagen; and 5) the enzyme specifically cleaves a -Pro-Gln- bond in the pro-alpha 1(I) chain and an -Ala-Gln- bond in the pro-alpha 2(I) chain. The bovine N-proteinase with a mass of 300 kDa was found to be similar to the 500-kDa enzyme and appeared to be a degraded form of the 500-kDa enzyme generated during purification. The N-proteinase from fetal bovine skin extracts also contained 300-kDa and 500-kDa enzyme forms.

Lymphatic transport of cholesterol from exogenous and biliary origins in nonfasting rats after intraduodenal infusions of triolein.

Labelled thoracic-duct lymph was collected from nonfasting rats with a bile fistula after simultaneous intraduodenal infusions of bile labelled with [1-2 3H] cholesterol and a nutritive mixture containing [4-14C] cholesterol. The gastrointestinal tract, feces, chylomicrons and infranatants were analysed. Both biliary and exogenous cholesterol were absorbed by lymphatic way but the recovery of 3H labelling in total lymph was markedly higher than that of 14C activity. This fact might be due to different rates of cholesterol exchanges from the two origins with the nonlabelled cholesterol present in the enterocytes and further exchanges of the enterocytes cholesterol with plasma cholesterol. Most of radioactivity was detected in chylomicrons. The relative [3H] and [14C] cholesterol specific activities were always low; thus when a little exogenous cholesterol is brought the major part of lymph cholesterol had an endogenous--other than biliary--source.