In vitro translation of chicken type X collagen in the presence of pancreas microsomes

Total RNA from epiphysis of 17-day-old chick embryo tibiae was used to direct protein synthesis in a wheat germ cell free system. The type X collagen chain, identified on the basis of its electrophoretic migration and of peptides obtained by S. aureus V8 protease digestion, was the major translation product. The newly synthesized chain included a signal sequence that was removed when dog pancreas membranes were added at the time of the protein synthesis.     

Macromolecular organization of collagen fibres in natural and tanned basement membrane

The elastic constants and ultrastructure of natural and tanned basement membrane of the crystalline lens of the adult rat have been investigated. Sonicated and negatively stained specimens of both membranes show parallel filaments that have similar spacing of 3.5(+/- 0.1) nm and a different periodicity. In natural membrane the periodicity is 3.7(+/- 0.13) nm, whilst in tanned basement membrane the periodicity is 3.2(+/- 0.15) nm. The periodicity ratio of tanned membrane to natural membrane was 0.86 +/- 0.04, whilst the elongation ratio of tanned membrane compared with natural membrane was 0.88 +/- 0.05. In contrast to this, the thickness ratio of tanned to natural membrane was 1.098 +/- 0.045. Tanned basement membrane showed a shrinkage of 12% in length but an increase in thickness of about 10%. These data suggest, firstly, that the degree of extension of the superhelices of the filaments follows closely the degree of extension of the intact membrane and, secondly, that the coiled superhelices of tanned membrane have an angle of tilt of about 42 degrees compared with those of natural membrane, where the angle is about 50 degrees. The Young's modulus of elasticity and ultimate stress of tanned basement membrane are, respectively, eight times greater and one-third as great as natural membrane. The entropy change in basement membrane was calculated from the external work necessary to extend the tanned membrane, and was estimated to be -13.5(+/- 2.4) J K-1 mol-1. An estimate of the change in entropy from thermodynamic measurements made on a suspension of collagen tanned with glutaraldehyde was found to be -30.1(+/- 9.5) J K-1 mol-1. The two different estimates of the change in entropy of collagen following tanning suggest that in basement membrane only about 45% of the collagenous protein has an extensile helical structure.     

Comparative studies of type X collagen expression in normal and rachitic chicken epiphyseal cartilage

The levels of type X collagen in mineralizing normal chicken epiphyses and nonmineralizing rachitic chicken tibial epiphyses were measured and compared. Qualitative immunoperoxidase studies with anti-chick type X collagen monoclonal antibodies on sections from normal and rachitic cartilage demonstrated that the type X collagen levels in rachitic growth plates are reduced. Northern hybridization of mRNA and biosynthetic studies have confirmed that type X collagen synthesis in rickets is also decreased. In hypocalcemic rickets, the level of type X collagen mRNA is reduced by 80% whereas the level of type X collagen mRNA is only reduced by 50% in normocalcemic rickets. These observations provide additional evidence that type X collagen is involved in the process of cartilage mineralization and also suggest that the partial recovery of type X collagen synthesis in normocalcemic rickets may be related to the elevated plasma concentration of calcium. Calcium concentration may therefore play an important role in the control of type X collagen synthesis.     

Macromolecular organization and in vitro growth characteristics of scaffold-free neocartilage grafts.

Recent advances in tissue engineering offer considerable promise for the repair of focal lesions in articular cartilage. Here we describe (1) the macromolecular organization of tissue-engineered neocartilage grafts at light and electron microscopic levels, (2) their in vitro development, and (3) the effect of chondrocyte dedifferentiation, induced by monolayer expansion, on their resultant structure. We show that grafts produced from primary cultures of chondrocytes are hyaline in appearance with identifiable zonal strata as evidenced by cell morphology, matrix organization, and immunohistochemical composition. Like native articular cartilage, their surface zone contains type I collagen, surface zone proteoglycan, biglycan and decorin with type II collagen, aggrecan, chondroitin sulfate, chondroitin-4-sulfate, and keratan sulfate, becoming more prominent with depth. Assessment of cell viability by Live/Dead staining and cell-cycle analysis with BrDU suggest that the in vitro tissue has a high cellular turnover and develops through both appositional and interstitial growth mechanisms. Meanwhile, cell-tracker studies with CMFDA (5-chloromethyl-fluorescein diacetate) demonstrate that cell sorting in vitro is not involved in their zonal organization. Finally, passage expansion of chondrocytes in monolayer culture causes progressive reductions in graft thickness, loss of zonal architecture, and a more fibrocartilaginous tissue histology, consistent with a dedifferentiating chondrocyte phenotype.     

The tissue form of chicken type VI collagen

We have purified intact type VI collagen from chicken gizzard. The protein was found to consist of a 130 kDa, a 140 kDa and a 180-200 kDa subunit. The 130 kDa and 140 kDa subunits were obtained in equimolar amounts and identified as the alpha 2 (VI) and the alpha 1 (VI) chains, respectively. The third subunit was usually obtained in the form of 3-4 closely related polypeptides, which may represent different processing or modification products of the alpha 3 (VI) chain.     

Partial characterization of type X collagen from bovine growth-plate cartilage. Evidence that type X collagen is processed in vivo

Sequential extraction of bovine growth-plate cartilage with 4 M guanidinium chloride and pepsin was used to identify the intact and pepsinized forms respectively of type X collagen. This collagen occurs predominantly as the processed [alpha 1(X)]3 form in vivo, although the procollagen [pro alpha 1(X)]3 form can also be detected. The bovine pro alpha 1(X) and alpha 1(X) chains have Mr values identical to the corresponding chick species (Mr 59,000 and 49,000). However, the pepsinized alpha 1(X)p chains (Mr 47,000) are larger than those of the chick (Mr 45,000), and the bovine collagen type X is further distinguished by being disulphide-bonded within the triple-helical domain.     

Immunoperoxidase localization of type X collagen in chick tibiae

Type X collagen was prepared from medium of long-term cultures of embryonic chick tibiotarsal chondrocytes. Antibodies to type X collagen were raised and used in immunoperoxidase localization studies with embryonic and growing chick tibiotarsus. Strong anti-type X collagen reactivity was detected mainly in the region of hypertrophic chondrocytes, and to a lesser extent in the zone of calcified cartilage. No reactivity was detected in the proliferative zone nor the superficial layer of the cartilage growth plate. These results suggest that type X collagen may play a key role in matrix calcification during growth and development of the skeletal system.     

Formation of collagen type I fibrils in vitro

The assembly of collagen fibrils as a function of temperature and collagen concentration was studied. It was shown that temperature increases from 25 to 35 degrees C, the degree of ordering of collagen fibrils increases 1.5-fold at collagen concentration above 1 mg/ml and 2-fold at low collagen concentration. A maximum ordering of fibril structure occurs under conditions close to physiological (T approximately 35 degrees C and collagen concentration 1.2 mg/ml). As temperature is elevated from 30 to 35 degrees C, the packing of collagen molecules in fibrils becomes more ordered: the values of enthalpy and entropy of the transition of fibrils from the native to a disordered state decrease at all collagen concentrations used. At high collagen concentration, the dimensions of cooperative blocks in fibrils formed at 25 and 30 degrees C coincide with those of cooperative blocks of monomeric collagen in solution. Upon increasing the temperature to 35 degrees C, the dimensions of cooperative blocks increase.     

Deposition of type X collagen in the cartilage extracellular matrix

In cultured chick embryo chondrocytes, type X collagen is preferentially deposited in the extracellular matrix, the ratio between type II and type X collagen being about 5 times higher in the culture medium than in the cell layer. When the newly synthesized collagens deposited in slices from the epiphyseal cartilage of 17-day-old embryo tibiae were isolated, type X collagen was always the major species. In agreement with this result the mRNA for type X collagen was the predominant mRNA species purified from the same tissue. When the total collagen (unlabeled) deposited in the epiphyseal cartilage was analyzed, it was observed that type X collagen represented only 1/15 of the type II collagen recovered in the same preparation. The possible explanations for these differences are discussed.     

Immunoelectron microscopic studies of type X collagen in endochondral ossification

Immunofluorescence and immunoelectron microscopy were used in conjunction with a monoclonal antibody to investigate the localization of type X collagen in the proximal tibial growth plate of 7-d-old chicks. This molecule was detected throughout the hypertrophic zone first appearing when chondrocytes exhibited hypertrophy: it was absent from the proliferative zone. Type X collagen was primarily associated with type II collagen fibrils as demonstrated by immunogold staining. Type X collagen was not concentrated in the focal calcification sites nor was it associated with matrix vesicles. These observations suggest that type X collagen may play a role other than that directly related to the nucleation of calcification.     

Macromolecular organization of bovine lens capsule

Rabbit antisera to type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin were used to localize these proteins in cross-sections of bovine anterior lens capsule. The antisera were exposed to (a) 10-micron frozen-thawed sections of formaldehyde-fixed tissue for examination in the light microscope by the indirect immunofluorescence method and (b) formaldehyde-fixed and L. R. White plastic-embedded thin sections for electron microscopic examination by the protein A-gold technique. The intensity of immunofluorescence was both uniform and strong throughout for type IV collagen, laminin and entactin, but patchy and weak for fibronectin. Electron microscopic immunolabeling with protein A-gold showed that all five components were distributed throughout the full thickness of the membrane, albeit the density of gold particles was not identical for all basement membrane proteins. In general, the number of particles per micron2 was greatest for type IV collagen and entactin, moderate for laminin and heparan sulfate proteoglycan and low for fibronectin. The ultrastructure of the lens capsule as examined by the electron microscope revealed a relatively uniform parallel alignment of filaments, thought to be collagenous. Since the distribution of the filaments corresponds well with the observed immunocytochemical pattern it is concluded that type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin co-localize throughout the cross-section of the anterior lens capsule.     

Cloning of the human and mouse type X collagen genes and mapping of the mouse type X collagen gene to chromosome 10.

Type X collagen, a homotrimer of alpha 1 (X) polypeptide chains, is specifically expressed by hypertrophic chondrocytes in regions of cartilage undergoing endochondral ossification. We have previously described the isolation of a small fragment of the human type X collagen gene (COL10A1) and its localization to the q21-q22 region of human chromosome 6 [Apte, S., Mattei, M.-G. & Olsen, B. R. (1991) FEBS Lett. 282, 393-396]. Using this fragment as a probe to screen genomic libraries, we report here the isolation of human and mouse genomic clones which contain the major part of the human and mouse type X collagen genes. In both species, the 14-kb genomic clones which were isolated contain a long open reading frame (greater than 2000 bp in length) which codes for the entire C-terminal non-collagenous (NC1) domain, the entire collagenous (COL) domain and part of the N-terminal non-collagenous (NC2) domain of the alpha 1(X) collagen chain. The human genomic clone contains the major part of the COL10A1 gene, in addition to the region we have previously cloned, and is highly similar to the corresponding portions of the mouse genomic clone (84.5% similarity at the nucleotide level, and 86.1% at the level of the conceptual translation product). The identification of the mouse genomic clone as the alpha 1(X) collagen gene (Col10a1) was confirmed by in situ hybridization of a fragment of the mouse genomic clone to sections from newborn mice. Hybridization was restricted to the hypertrophic chondrocytes of developing chondroepiphyses, being absent in small chondrocytes and in other tissues. Using interspecific backcross analysis, the locus for the mouse alpha 1 (X) collagen gene was assigned to chromosome 10. The cloning and chromosomal mapping of the human and mouse alpha 1 (X) collagen genes now permit the investigation of the possible role of type X collagen gene defects in the genesis of chondrodysplasias in both species and provide data essential for the generation of transgenic mice deficient in type X collagen.     

Developmental acquisition of type X collagen in the embryonic chick tibiotarsus

The temporal and spatial distribution of short chain skeletal (Type X) collagen was immunohistochemically examined in the chick tibiotarsus from 6 days of embryonic development to 1 day posthatching. The monoclonal antibody employed (AC9) was recently produced and characterized as being specific for an epitope located within the helical domain of the type X collagen molecule (T. M. Schmid and T. F. Linsenmayer, J. Cell Biol., in press). The earliest detectable appearance of type X collagen was at 7.5 days, at which time it was restricted to a middiaphyseal location (i.e., in the primary center of ossification). This was in marked contrast to type II collagen, which appears earlier and is distributed throughout the cartilaginous anlagen. With increasing embryonic age, the reactivity with the type X antibody progressively extended toward the epiphyses, lagging somewhat behind the progression of chondrocyte hypertrophy. The anti-type X collagen antibody also reacted with the bony matrix itself, but the immunofluorescent signal produced by this source was considerably less than that produced by cartilage. At 19 days of development, a new small site of type X deposition was initiated in an epiphyseal location, which subsequently enlarged in circumference. These results are consistent with our previous biochemical studies suggesting that, in cartilage, type X collagen is specifically a product of that population of chondrocytes which have undergone hypertrophy.     

Macromolecular structure and organization of alpha keratin

The small-angle equatorial X-ray scattering of alpha keratin (African porcupine quill) was studied using a direct type of analysis, which allows the separation of the intermicro-fibrillar interference effects from those of the intramicrofibrillar scattering without the use of prior assumptions. X-ray data were obtained from 470–21 Å; their analysis by the above method resulted in a z-axis projection of the microfibrillar electron density, which extends over a diameter of 80 Å and has a core region surrounded by a peak located at a radius of 28 Å. The macromolecular organization of the 80-Å-diameter microfibrils was found to be an arc-like layer type of aggregation and not a pseudohexagonal packing. When the specimen was subjected to silver staining the aggregation of the microfibrils did not change except for a slight contraction. The z-axis projection of the stained microfibril, however, was significantly different. The silver appears to build up slightly around the outer periphery of the microfibril, stains the peripheral region very little, deposits on the inside of the peripheral region, and outlines the core area.     

Cell hypertrophy and type X collagen synthesis in cultured articular chondrocytes

Articular cartilage is a permanent tissue whose cells do not normally take part in the endochondral ossification process. To determine whether articular chondrocytes possess the potential to express traits associated with this process such as cell hypertrophy and type X collagen, chondrocytes were isolated from adult chicken tibial articular cartilage and maintained in long-term suspension cultures. As a positive control in these experiments, we used parallel cultures of chondrocytes from the caudal portion of chick embryo sternum. Both articular and sternal chondrocytes readily proliferated and progressively increased in size with time in culture. Many had undergone hypertrophy by 4-5 weeks. Analysis of medium-released collagenous proteins revealed that both articular and sternal chondrocytes initiated type X collagen synthesis between 3 and 4 weeks of culture; synthesis of this macromolecule increased with further growth. Immunofluorescence analysis of 5-week-old cultures showed that about 15% of articular chondrocytes and 30% of sternal chondrocytes produced type X collagen; strikingly, there appeared to be no obvious relationship between type X collagen production and cell size. The results of this study show that articular chondrocytes from adult chicken tibia possess the ability to express traits associated with endochondral ossification when exposed to a permissive environment. They suggest also that the process of cell hypertrophy and initiation of type X collagen synthesis are independently regulated both in articular and sternal chondrocytes.     

Molecular organization of type IV collagen: polymer liquid crystal-like aspects

A new X-ray diffraction pattern from type IV collagen is described, which can be interpreted on the basis of crystalline and liquid crystalline origins of the reflections. Bovine anterior lens capsules extracted with 1 NaCl and oriented by extension of 60% under constant load gave medium angle X-ray diffraction patterns showing many of the characteristics typical of liquid crystals. Prominent features, apart from those wide angle features attributable to the collagen triple helix, are (1) a four-point pattern of broad reflections at d-spacing 3.9 nm, and layer line spacing near 5 nm. (2) A broad intense equatorial peak centred at 1.24 nm, indicative of LIQUID=like lateral molecular associations (3) A set of five sharp, streaked meridional reflections (previously obscured by the broad peak near 5 nm in unextracted capsules). (4) A further six higher angle reflections of a diffuse, arced and broad appearance on the meridian. The sharp streaked meridional reflections emanate from a long-range periodicity of units 8–9 nm in diameter. These features form a self-consistent system if interpreted on the basis of staggered liquid crystal-like array of collagen molecules, in which case the first five meridionals and remaining broad reflections, sampled on the meridian, can all be indexed as orders of 21 nm.     

Molecular assembly of recombinant chicken type II collagen in the yeast Pichia pastoris

Effective treatment of rheumatoid arthritis can be mediated by native chicken type II collagen(n CCII), recombinant peptide containing n CCII tolerogenic epitopes(CTEs), or a therapeutic DNA vaccine encoding the full-length CCOL2 A1 c DNA. As recombinant CCII(r CCII) might avoid potential pathogenic virus contamination during n CCII preparation or chromosomal integration and oncogene activation associated with DNA vaccines, here we evaluated the importance of propeptide and telopeptide domains on r CCII triple helix molecular assembly. We constructed p C-and p N-procollagen(without N-or Cpropeptides, respectively) as well as CTEs located in the triple helical domain lacking both propeptides and telopeptides, and expressed these in yeast Pichia pastoris host strain GS115(his4, Mut+) simultaneously with recombinant chicken prolyl-4-hydroxylase α and β subunits. Both p C-and p N-procollagen monomers accumulated inside P. pastoris cells, whereas CTE was assembled into homotrimers with stable conformation and secreted into the supernatants, suggesting that the large molecular weight p C-or p N-procollagens were retained within the endoplasmic reticulum whereas the smaller CTEs proceeded through the secretory pathway. Furthermore, resulting recombinant chicken type II collagen p Cα1(II) can induce collagen-induced arthritis(CIA) rat model, which seems to be as effective as the current standard n CCII. Notably, protease digestion assays showed that r CCII could assemble in the absence of C-and N-propeptides or telopeptides. These findings provide new insights into the minimal structural requirements for r CCII expression and folding.