N P Morris

Ultrastructural localization of collagen types II, IX, and XI in the growth plate of human rib and fetal bovine epiphyseal cartilage: type XI collagen is restricted to thin fibrils.

The collagen fibrils of hyaline cartilage vary in diameter depending on developmental stage and location within the tissue. In general, growth plates and fetal epiphyseal cartilages contain fibrils with diameters of less than approximately 25 nm, whereas the permanent cartilage of adult tissues contains fibrils of approximately 30-200 nm. The interstitial collagen fibrils of fetal cartilage are complex, having at least three collagen types as integral components. Type XI, a member of the fibrillar collagen class, has been proposed to limit fibril diameter. To test this proposition we sought to determine if Type XI collagen was preferentially associated with fibrils of smaller diameter. We focused our study on human juvenile rib growth plate, which has thin fibrils in the hypertrophic zone, thick fibrils in the resting zone or permanent cartilage, and a mixture of thin and thick fibrils in the proliferative zone. Tissues were examined by immunoelectron microscopy with antipeptide antibodies to the carboxyl telopeptide and to the amino terminal non-triple-helical domains of alpha 1 (XI). These studies showed that (a) both epitopes of Type XI collagen were readily accessible to antibodies at the fibrillar surface, (b) Type XI collagen was associated predominantly with fibrils < 25 nm in diameter, (c) Type XI collagen was not found in thick fibrils even after disruption with chaotropic agents, and (d) collagen Types II and IX were associated with fibrils of all sizes. These studies were extended to human newborn epiphyseal cartilage and to fetal calf cartilage, with the same result.

Developmentally Regulated Alternative Splicing of the α1(XI) Collagen Chain: Spatial and Temporal Segregation of Isoforms in the Cartilage of Fetal Rat Long Bones

Type XI collagen is a component of the heterotypic collagen fibrils of fetal cartilage and is required to maintain the unusually thin diameter of these fibrils. The mature matrix form of the molecule retains an N-terminal variable region whose structure is modulated by alternative exon splicing that is tissue-specific and developmentally regulated. In the α1(XI) chain, antibodies to two of the peptides, p6b and p8, encoded by the alternatively spliced exons localized these epitopes to the surface of the collagen fibrils and were used to determine the pattern of isoform expression during the development of rat long bones (humerus). Expression of the p6b isoform was restricted to the periphery of the cartilage underlying the perichondrium of the diaphysis, a pattern that appears de novo at embryonic Day (E) 14. P8 isoforms appeared to be associated with early stages of chondrocyte differentiation and were detected throughout prechondrogenic mesenchyme and immature cartilage. After E16, p8 isoforms gradually disappeared from the diaphysis and then from the epiphysis preceding chondrocyte hypertrophy, but were highly evident at the periarticular joint surface, where ongoing chondrogenesis accompanies the formation of articular cartilage. The spatially restricted and differentiation-specific distribution of α1(XI) isoforms is evidence that Type XI collagen participates in skeletal development via a mechanism that may be distinct from regulation of fibrillogenesis.

Analysis of the Thermal Stability of Type II Collagen in Various Solvents Used for Reversed-Phase High Performance Chromatography

The thermal stability of type II collagen in various solvents is shown to depend on the ability of the solvent to form hydrogen bonds. Mixtures of water with 1-propanol, 2-propanol, 1,2-propanediol, tetrahydrofuran and acetonitrile effect the stability of the triple helix differently. The temperature of the triple helix coil transition of type II collagen in 50(v/v)% solvent mixture in 0.1% trifluoroacetic acid ranges from 34 degrees C for 1,2-propanediol to 22.5 degrees C for acetonitrile, compared to 38 degrees C in 0.1% trifluoroacetic acid and 41.5 degrees C at neutral pH. There is no correlation between the dielectric constants of the solvents and the decrease in thermal stability, indicating that electrostatic interactions play only a minor role in the stability of the triple helix. Acetonitrile and tetrahydrofuran destabilize the triple helix more than the solvents containing hydroxyl groups. For reversed-phase high performance chromatography 2-propanol is the solvent of choice, but temperature control is very important, because the interaction of the triple helix with the column matrix leads to an additional destabilization of the triple helix beyond the destabilization effect of the solvent. In acetonitrile, a solvent commonly used for reversed-phase high performance chromatography, the triple helix is completely denatured when eluted from a C18 column at room temperature.

Characterization of EHD4, an EH domain-containing protein expressed in the extracellular matrix.

To identify proteins that promote assembly of type VI collagen tetramers or stabilize type VI collagen filaments, a two-hybrid screen of a human placenta library was used and a new extracellular protein discovered. The cDNA sequence of the new protein encodes 541 amino acid residues. This cDNA sequence is identical to EHD4, a recently described member of the EH domain family of proteins. Two mRNAs of 4.4 and 3.0 kilobases were present in human skin fibroblasts and most tissues tested but were most prevalent in the heart. The chromosomal localization of the gene for this new protein was determined to be at 15q14-q15. Three polyclonal peptide antibodies were made against synthetic EHD4 peptides. The affinity-purified antibodies were used in immunofluorescent staining of developing limbs and matrices produced by human skin fibroblasts and mouse NIH3T3 fibroblasts in culture. Embryonic rat limb cartilage was strongly stained throughout development, and cultured fibroblasts deposited an extracellular filamentous network containing EHD4. In non-denaturing extracts of fetal bovine cartilage and in human skin fibroblast culture media, two components of ∼220 and 158 kDa were observed, which, after reduction, migrated as a 56-kDa component on SDS-polyacrylamide gel electrophoresis. EHD4 is the first extracellular matrix protein described that contains an EH domain.

Cis-acting elements regulate alternative splicing of exons 6A, 6B and 8 of the α1(XI) collagen gene and contribute to the regional diversification of collagen XI matrices

Consecutive exons 6A, 6B, 7 and 8 that encode the variable region of the amino-terminal domain (NTD) of the col11a1 gene product undergo a complex pattern of alternative splicing that is both tissue-dependent and developmentally regulated. Expression of col11a1 is predominantly associated with cartilage where it plays a critical role in skeletal development. At least five splice-forms (6B-7-8, 6A-7-8, 7-8, 6B-7 and 7) are found in cartilage. Splice-forms containing exon 6B or 8 have distinct distributions in the long bone during development, while in non-cartilage tissues, splice-form 6A-7-8 is typically expressed. In order to study this complex and tissue-specific alternative splicing, a mini-gene that contains mouse genomic sequence from exon 5 to 11, flanking the variable region of alpha1(XI)-NTD, was constructed. The minigene was transfected into chondrocytic (RCS) and non-chondrocytic (A204) cell lines that endogenously express alpha1(XI), as well as 293 cells which do not express alpha1(XI). Alternative splicing in RCS and A204 cells reflected the appropriate cartilage and non-cartilage patterns while 293 cells produced only 6A-7-8. This suggests that 6A-7-8 is the default splicing pathway and that cell or tissue-specific trans-acting factors are required to obtain pattern of the alternative splicing of alpha1(XI) pre-mRNA observed in chondrocytes. Deletional analysis was used to identify cis-acting regions important for regulating splicing. The presence of the intact exon 7 was required to generate the full complex chondrocytic pattern of splicing. Furthermore, deletional mapping of exon 6B identified sequences required for expression of exon 6B in RCS cells and these may correspond to purine-rich (ESE) and AC-rich (ACE) exonic splicing enhancers.

DISTRIBUTION OF COLLAGEN XII AND XIV IN THE BLADDER WALL OF THE MALE RAT WITH OUTLET OBSTRUCTION

PURPOSE Our previous studies indicate that neonatal estrogenization with diethylstilbestrol (neoDES) of male mice and rats causes partial outlet obstruction. In the present study, type XII and XIV collagens were localized in the bladder to study their role in the development of obstruction. MATERIALS AND METHODS The bladder sections immunostained with smooth muscle specific a-actin antibody were double labeled either with collagen type XII or type XIV antibodies. The specimens were then analyzed with conventional and confocal fluorescence microscope. RESULTS Type XII and XIV collagens were not evenly distributed in the bladder. Further, in neonatally estrogenized rats collagen XIV appeared inside smooth muscle fascicles. CONCLUSIONS Non-overlapping distributions of collagen XII and XIV suggest their different roles in the urinary bladder. Penetration of collagen XIV inside smooth muscle fascicles may have a role in the development of DES-induced partial outlet obstruction.

Production and Characterization of Hybridoma Antibody to Native Human Type II Collagen

Using a sensitive RIA for collagenous proteins, we have applied a series of consecutive selection steps to construct a hybridoma that produces antibody against native human Type II collagen. The selection steps consisted of: 1) hybrid cell line selection; 2) co-selection of IgG class immunoglobulin reactive to the immunizing antigen; 3) selection for anticollagen antibody by complete inhibition of antibody binding to Type II collagen-coated plates by prior specific collagenase digestion; 4) selection for anti-helical antibody by complete inhibition of antibody binding to heat-denatured Type II collagen-coated plates; 5) clonal selection; 6) selection for absence of crossreactivity to 5-7 additional native collagen types; and finally 7) selection for antibody yielding indirect immunofluorescent staining of human cartilage matrix, but not adjacent bone or perichondrium. Cross-reactivity assays against other native human collagen types revealed little cross-reactivity. In addition, we have demonstrated that native Type II collagen inhibits antibody binding to Type II collagen-coated plates; that Type II collagen can be recovered from antigen-antibody complexes; that a sensitive radioimmunoassay for Type II collagen can be developed; and that the antibody is useful for indirect immunofluorescent studies of human cartilage matrix.

Thermal Stability and Folding of the Triple Helices of Interstitial and Basement Membrane Collagens

Vertebrate collagens comprise a family of extracellular matrix proteins which form fibrils or networks containing one type (homotypic) or multiple types (heterotypic) of molecules. Each of the twelve genetically distinct types of collagen contains multiple structural domains. By definition, one or more of these domains is the unique collagen triple helix. The collagen structural motif can be generalized as three colinear polypeptides forming a central triple helix flanked at both the amino and carboxyl ends by non- helical domains. Where these flanking regions are subsequently removed by specific proteolysis, the parent molecule is called a procollagen. The triple helix then provides an extended conformation which forms higher order aggregates through lateral associations. In some cases, end to end interactions are mediated through retained non helical domains flanking the helix.