scholarly journals Alternatively spliced type II procollagen mRNAs define distinct populations of cells during vertebral development: differential expression of the amino-propeptide.

1991 ◽  
Vol 114 (6) ◽  
pp. 1307-1319 ◽  
Author(s):  
L J Sandell ◽  
N Morris ◽  
J R Robbins ◽  
M B Goldring
Keyword(s):  
Differential Expression ◽  
Structural Integrity ◽  
Type Ii Collagen ◽  
Nerve Tissue ◽  
Tissue Type ◽  
Collagen Molecule ◽  
Gene Transcript ◽  
Functional Difference ◽  
Type Ii ◽  
Exon 2

Type II collagen is a major component of cartilage providing structural integrity to the tissue. Type II procollagen can be expressed in two forms by differential splicing of the primary gene transcript. The two mRNAs either include (type IIA) or exclude (type IIB) an exon (exon 2) encoding the major portion of the amino (NH2)-propeptide (Ryan, M. C., and L. J. Sandell. 1990. J. Biol. Chem. 265:10334-10339). The expression of the two procollagens was examined in order to establish a potential functional significance for the two type II procollagen mRNAs. First, to establish whether the two mRNAs are functional, we showed that both mRNAs can be translated and the proteins secreted into the extracellular environment. Both proteins were identified as type II procollagens. Secondly, to test the hypothesis that differential expression of type II procollagens may be a marker for a distinct population of cells, specific procollagen mRNAs were localized in tissue by in situ hybridization to oligonucleotides spanning the exon junctions. Embryonic vertebral column was chosen as a source of tissue undergoing rapid chondrogenesis, allowing the examination of a variety of cell types related to cartilage. In this issue, each procollagen mRNA had a distinct tissue distribution during chondrogenesis with type IIB expressed in chondrocytes and type IIA expressed in cells surrounding cartilage in prechondrocytes. The morphology of the cells expressing the two collagen types was distinct: the cells expressing type IIA are narrow, elongated, and "fibroblastic" in appearance while the cells expressing type IIB are large and round. The expression of type IIB appears to be correlated with abundant synthesis and accumulation of cartilagenous extracellular matrix. The expression of type IIB is spatially correlated with the high level expression of the cartilage proteoglycan, aggrecan, establishing type IIB procollagen and aggrecan as markers for the chondrocyte phenotype. Transcripts of type II collagen, primarily type IIA, are also expressed in embryonic spinal ganglion. While small amounts of type II collagen have been previously detected in noncartilagenous tissues, the detection of this new form of the collagen in relatively high abundance in embryonic nerve tissue is unique. Taken together, these findings imply a potential functional difference between type IIA and type IIB procollagens and indicate that the removal of exon 2 from the pre-mRNA, and consequently the NH2-propeptide from the collagen molecule, may be an important step in chondrogenesis. In addition, type II procollagen, specifically type IIA, may function in noncartilage tissues, particularly during development.

scholarly journals Type IIA Procollagen Amino Propeptide Is Localized in Human Embryonic Tissues

1997 ◽  
Vol 45 (11) ◽  
pp. 1469-1480 ◽  
Author(s):  
Anush Oganesian ◽  
Yong Zhu ◽  
Linda J. Sandell
Keyword(s):  
Extracellular Matrix ◽  
Type Ii Collagen ◽  
Protein Domain ◽  
Collagen Molecule ◽  
Endochondral Bone Formation ◽  
Type Ii ◽  
Exon 2 ◽  
Helical Domain ◽  
Chondroprogenitor Cells ◽  
Triple Helical Domain

Type II procollagen is synthesized in two forms generated by the alternative splicing of its precursor mRNA. The alternatively spliced domain, exon 2, encodes the 69-amino-acid cysteinerich region of the NH2 propeptide. Studies of mRNA expression have shown that the longer form, designated Type IIA procollagen, is synthesized by chondroprogenitor cells and various noncartilaginous tissues. The shorter form, Type IIB procollagen, is synthesized by differentiated chondrocytes. As the initial step in our investigations of the function of the Type IIA procollagen, the protein domain corresponding to exon 2 was created as a recombinant fusion protein and used to raise antibodies in rabbits. The resulting antiserum was specific for Type IIA procollagen NH2 propeptide as shown by ELISA, Western blotting, and immunofluorescent co-localization with the triple-helical domain of Type II collagen. Type IIA procollagen was identified in tissue culture medium of 54-day human fetal ribs. Confocal microscopy was used to localize the Type IIA NH2 propeptide in Day 50 and 53 human embryos. In the digital rays of the developing hand, where only Type IIA procollagen mRNA was detected, Type IIA procollagen NH2 propeptide was observed in the extracellular matrix. The presence of Type IIA procollagen NH2 propeptide was observed in the cartilage of the developing long bones of the lower arm and vertebral bodies even though these tissues synthesize Type IIB mRNA at this developmental stage. Type IIA procollagen NH2 propeptide was localized in the developing trachea, a cartilage that does not undergo endochondral bone formation. Type IIA NH2 propeptide was also localized in noncartilaginous tissues known to synthesize Type IIA mRNA, such as the intervertebral area, perichondrium, notochordal sheath, and neuroepithelium of the otic vesicle. In most tissues, co-localization with antiserum against the triple-helical domain of Type II collagen was observed. Positive immunoreactivity with the Type IIA NH2 propeptide antiserum indicates, for the first time, that this propeptide is present in the tissue. Co-localization of NH2 propeptide antibodies with the triple-helical domain of the collagen molecule suggests that Type IIA procollagen is intact in the extracellular matrix of these tissues. Taken together, these results strongly suggest that around cells that synthesize Type IIA procollagen mRNA, Type IIA procollagen NH2 propeptide is secreted and deposited into the extracellular matrix. In light of these results, we predict that Type IIA procollagen plays a role in differentiation of tissues that augments its purely architectural function. (J Histochem Cytochem 45:1469–1480, 1997)

scholarly journals Chondrodysplasia in transgenic mice harboring a 15-amino acid deletion in the triple helical domain of pro alpha 1(II) collagen chain.

1992 ◽  
Vol 118 (1) ◽  
pp. 203-212 ◽  
Author(s):  
M Metsäranta ◽  
S Garofalo ◽  
G Decker ◽  
M Rintala ◽  
B de Crombrugghe ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Endochondral Ossification ◽  
Electron Microscopic Examination ◽  
Type Ii Collagen ◽  
Dominant Negative ◽  
Collagen Molecule ◽  
Type Ii ◽  
Electron Microscopic ◽  
Dominant Negative Mutation ◽  
Collagen Chain

We have generated transgenic mice by microinjection of a 39-kb mouse pro alpha 1(II) collagen gene construct containing a deletion of exon 7 and intron 7. This mutation was expected to disturb the assembly and processing of the homotrimeric type II collagen molecule in cartilage. Expression of transgene mRNA at levels equivalent or higher than the endogenous mRNA in the offspring of two founder animals resulted in a severe chondrodysplastic phenotype with short limbs, hypoplastic thorax, abnormal craniofacial development, and other skeletal deformities. The affected pups died at birth due to respiratory distress. Light microscopy of epiphyseal growth plates of transgenic pups demonstrated a marked reduction in cartilaginous extracellular matrix and disruption of the normal organization of the growth plate. The zone of proliferating chondrocytes was greatly reduced whereas the zone of hypertrophic chondrocytes was markedly increased extending deep into the diaphysis suggestive of a defect in endochondral ossification. Electron microscopic examination revealed chondrocytes with extended RER, a very severe reduction in the amount of cartilage collagen fibrils, and abnormalities in their structure. We postulate that the deletion in the alpha 1(II) collagen acts as a dominant negative mutation disrupting the assembly and secretion of type II collagen molecules. The consequences of the mutation include interference with normal endochondral ossification. These mice constitute a valuable model to study the mechanisms underlying human chondrodysplasias and normal bone formation.

scholarly journals Localization of an arthritogenic epitope to a fragment of the type II collagen molecule.

Ensho ◽  
1986 ◽  
Vol 6 (4) ◽  
pp. 351-356
Author(s):  
Kuniaki Terato ◽  
Yasunori Shimozuru ◽  
Karen A. Hasty ◽  
Michael A. Cremer ◽  
John M. Stuart ◽  
...  
Keyword(s):  
Type Ii Collagen ◽  
Collagen Molecule ◽  
Type Ii

scholarly journals Substitution of aspartic acid for glycine at position 310 in type II collagen produces achondrogenesis II, and substitution of serine at position 805 produces hypochondrogenesis: analysis of genotype-phenotype relationships

1995 ◽  
Vol 307 (3) ◽  
pp. 823-830 ◽  
Author(s):  
J Bonaventure ◽  
L Cohen-Solal ◽  
P Ritvaniemi ◽  
L Van Maldergem ◽  
N Kadhom ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Type Ii Collagen ◽  
Terminal Region ◽  
Collagen Molecule ◽  
Type I ◽  
Type Ii ◽  
Single Base ◽  
Glycine Substitution ◽  
Achondrogenesis Type

Two different mutations were found in two unrelated probands with lethal chondrodysplasias, one with achondrogenesis type II and the other with the less severe phenotype of hypochondrogenesis. The mutations in the COL2A1 gene were identified by denaturing gradient gel electrophoresis analysis of genomic DNA followed by dideoxynucleotide sequencing and restriction site analysis. The proband with achondrogenesis type II had a heterozygous single-base mutation that substituted aspartate for glycine at position 310 of the alpha 1(II) chain of type II procollagen. The proband with hypochondrogenesis had a heterozygous single-base mutation that substituted serine for glycine at position 805. Type II collagen extracted from cartilage from the probands demonstrated the presence of type I collagen and a delayed electrophoretic mobility, indicating post-translational overmodifications. Analysis of CNBr peptides showed that, in proband 1, the entire peptides were overmodified. Examination of chondrocytes cultured in agarose or alginate indicated that there was a delayed secretion of type II procollagen. In addition, type II collagen synthesized by cartilage fragments from the probands demonstrated a decreased thermal stability. The melting temperature of the type II collagen containing the aspartate-for-glycine substitution was reduced by 4 degrees C, and that of the collagen containing the serine-for-glycine substitution was reduced by 2 degrees C. Electron microscopy of the extracellular matrix from the chondrocyte cultures showed a decreased density of matrix and the presence of unusually short and thin fibrils. Our results indicate that glycine substitutions in the N-terminal region of the type II collagen molecule can produce more severe phenotypes than mutations in the C-terminal region. The aspartate-for-glycine substitution at position 310, which was associated with defective secretion and a probable increased degradation of collagen, is the most destabilizing mutation yet reported in type II procollagen.

scholarly journals Collagen-induced arthritis in mice. Localization of an arthritogenic determinant to a fragment of the type II collagen molecule.

1985 ◽  
Vol 162 (2) ◽  
pp. 637-646 ◽  
Author(s):  
K Terato ◽  
K A Hasty ◽  
M A Cremer ◽  
J M Stuart ◽  
A S Townes ◽  
...  
Keyword(s):  
Cyanogen Bromide ◽  
Type Ii Collagen ◽  
The Other ◽  
Collagen Molecule ◽  
Strong Positive Correlation ◽  
Type Ii ◽  
Collagen Induced Arthritis ◽  
Positive Correlation

Purified chick type II collagen was cleaved with cyanogen bromide (CB), and the resulting peptides isolated and renatured. Sera from arthritic DBA/1 mice, immunized with chick type II collagen, were tested for reactivity with each peptide. The sera preferentially recognized peptides 11, 10, and 8, in that order. Some reactivity was also detected to peptides 9, 7, and 12. Because arthritis depends upon binding of antibody to autologous type II collagen in the joint, sera were also tested for reactivity with mouse type II collagen. There was a strong positive correlation between reactivity with peptide 11 and reactivity with mouse collagen, but no correlation was found with any of the other peptides. Peptides 11, 10, and 8 were also used for immunization. Antibodies were detected in response to each of these peptides, but arthritis developed only in mice immunized with peptide 11. We conclude that a major immunogenic and arthritogenic epitope on type II collagen resides in the region of the molecule represented by CB peptide 11.

scholarly journals Murine fibromodulin: cDNA and genomic structure, and age-related expression and distribution in the knee joint

2001 ◽  
Vol 355 (3) ◽  
pp. 577-585 ◽  
Author(s):  
Anna-Marja K. SÄÄMÄNEN ◽  
Heli J. SALMINEN ◽  
A. Juho RANTAKOKKO ◽  
Dick HEINEGÅRD ◽  
Eero I. VUORIO
Keyword(s):  
Genomic Structure ◽  
Type Ii Collagen ◽  
Type I ◽  
Mrna Levels ◽  
Primary Role ◽  
Type Ii ◽  
Exon 2 ◽  
Collagen Fibrillogenesis ◽  
Calcified Cartilage ◽  
Age Related

The genomic structure of murine fibromodulin was determined, and its age-related expression and distribution were characterized in knee epiphyses, with decorin studied for reference. Fibromodulin, as well as decorin, have roles in collagen fibrillogenesis both in vitro and in vivo. The murine fibromodulin gene, Fmod, was similar with that in other species, with three exons and 86% of the translated sequence in exon 2. The 2.7kb long cDNA contains an open reading frame of 1131nt. Fibromodulin mRNA levels were highest in tissues rich in fibrillar collagens type I or type II. During growth, the distribution of fibromodulin mRNA was similar with that of type II collagen, with the highest levels between 5 days and 1 month of age. Thereafter, the expression of type II collagen declined to a level near the detection limit, whereas the fibromodulin expression decreased less markedly to a level of approx. 35% of maximum, and remained constant throughout the rest of the observation period. In contrast, decorin mRNA levels were the highest in old animals. Pericellular deposition of fibromodulin was strong around the late-hypertrophic chondrocytes of the secondary ossification centre and in the growth plate. In young epiphyses, both fibromodulin and decorin were found interterritorially, mainly in the uncalcified and deep-calcified cartilage. In the old mice, calcified cartilage became enriched with regard to fibromodulin, while, in contrast, decorin deposition diminished, particularly near the tidemark. In the subchondral bone trabeculae, decorin was found in the endosteum of growing, but not in the mature, epiphyses. Differences in the expression and distribution profiles suggest different roles for fibromodulin and decorin in the regulation of collagen fibrillogenesis, maintenance of the fibril organization and matrix mineralization. As fibromodulin is deposited closer to cells than decorin, it may have a primary role in collagen fibrillogenesis, whereas decorin might be involved in the maintenance of fibril structures in the interterritorial matrix.

Expression of type II procollagens during development of the human intervertebral disc

2002 ◽  
Vol 30 (6) ◽  
pp. 831-838 ◽  
Author(s):  
A. McAlinden ◽  
Y. Zhu ◽  
L. J. Sandell
Keyword(s):  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Type Ii Collagen ◽  
Intervertebral Discs ◽  
Bone Morphogenetic Protein 2 ◽  
Type Ii ◽  
Exon 2 ◽  
Morphogenetic Protein ◽  
Alternatively Spliced ◽  
Specific Stage

Mice lacking type II collagen fail to develop intervertebral discs. The present study describes the distribution of the developmentally expressed type IIA procollagen molecule, as well as types I and III collagens, in human IV disc specimens ranging from 42 to 101 days gestation. Type IIA procollagen contains the alternatively spliced exon 2 which encodes a 69-amino-acid cysteinerich domain. By radioactive in situ hybridization and fluorescence immunohistochemistry, we identified changes in the localization patterns of type IIA procollagen, particularly between days 54 and 101. At day 54, the developing disc was divided into the outer annulus containing types I and III collagens, the inner annulus containing type IIA procollagen and the notochord consisting of all three fibrillar collagens. Specifically, the IIA N-terminal propeptide was localized in the extracellular matrix at day 54 but, by day 101, was only observed in the cytoplasm of the inner annulus cells. A functional role for the IIA N-terminal propeptide during this specific stage of disc development seems apparent. This function may involve regulation of growth factors since the exon 2-encoded domain of type IIA procollagen has previously been shown to bind to bone morphogenetic protein-2 and transforming growth factor-β. We aim to explore this mechanism further.

scholarly journals Effects of ovariectomy and estrogen therapy on type II collagen degradation and structural integrity of articular cartilage in rats: Implications of the time of initiation

2006 ◽  
Vol 54 (8) ◽  
pp. 2441-2451 ◽  
Author(s):  
Svetlana Oestergaard ◽  
Bodil C. Sondergaard ◽  
Pernille Hoegh-Andersen ◽  
Kim Henriksen ◽  
Per Qvist ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Structural Integrity ◽  
Estrogen Therapy ◽  
Type Ii Collagen ◽  
Collagen Degradation ◽  
Type Ii

C-propeptide of type ii procollagen; a protein associated with cartilage mineralization

1986 ◽  
Vol 44 ◽  
pp. 272-273
Author(s):  
E.R. Lee ◽  
A.R. Poole
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Matrix Protein ◽  
Type Ii Collagen ◽  
Collagen Molecule ◽  
Type Ii ◽  
Amino Acid Sequencing ◽  
Procollagen Type ◽  
Matrix Distribution ◽  
The Matrix

A matrix protein has been identified in both calcifying and noncalcifying cartilage during development, and named chondrocalcin. The concentration of this protein is greatly increased in calcifying cartilage and it appears in the matrix when and where mineralization occurs. Amino acid sequencing has recently shown that chondrocalcin is identical to the C-propeptide of type II procollagen. Type II procollagen is a high molecular weight precursor of type II collagen and it is characterized by amino (NH2) and carboxy (C) propeptide extensions. These nonhelical extensions are normally cleaved extracellularly by proteinases to give the collagen molecule. To investigate the synthesis, secretion and matrix distribution of the C-propeptide, particularly during mineralization, this protein has been localized at the EM level with immunogold techniques.

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