Shi-Wu Li

Human Ehlers-Danlos Syndrome Type VII C and Bovine Dermatosparaxis Are Caused by Mutations in the Procollagen I N-Proteinase Gene

Ehlers-Danlos syndrome (EDS) type VIIC is a recessively inherited connective-tissue disorder, characterized by extreme skin fragility, characteristic facies, joint laxity, droopy skin, umbilical hernia, and blue sclera. Like the animal model dermatosparaxis, EDS type VIIC results from the absence of activity of procollagen I N-proteinase (pNPI), the enzyme that excises the N-propeptide of type I and type II procollagens. The pNPI enzyme is a metalloproteinase containing properdin repeats and a cysteine-rich domain with similarities to the disintegrin domain of reprolysins. We used bovine cDNA to isolate human pNPI. The human enzyme exists in two forms: a long version similar to the bovine enzyme and a short version that contains the Zn++-binding catalytic site but lacks the entire C-terminal domain in which the properdin repeats are located. We have identified the mutations that cause EDS type VIIC in the six known affected human individuals and also in one strain of dermatosparactic calf. Five of the individuals with EDS type VIIC were homozygous for a C-->T transition that results in a premature termination codon, Q225X. Four of these five patients were homozygous at three downstream polymorphic sites. The sixth patient was homozygous for a different transition that results in a premature termination codon, W795X. In the dermatosparactic calf, the mutation is a 17-bp deletion that changes the reading frame of the message. These data provide direct evidence that EDS type VIIC and dermatosparaxis result from mutations in the pNPI gene.

Mutations in COL11A2 cause non-syndromic hearing loss (DFNA13)

We report that mutation of COL11A2 causes deafness previously mapped to the DFNA13 locus on chromosome 6p. We found two families (one American and one Dutch) with autosomal dominant, non-syndromic hearing loss to have mutations in COL11A2 that are predicted to affect the triple-helix domain of the collagen protein. In both families, deafness is non-progressive and predominantly affects middle frequencies. Mice with a targeted disruption of Col11a2 also were shown to have hearing loss. Electron microscopy of the tectorial membrane of these mice revealed loss of organization of the collagen fibrils. Our findings revealed a unique ultrastructural malformation of inner-ear architecture associated with non-syndromic hearing loss, and suggest that tectorial membrane abnormalities may be one aetiology of sensorineural hearing loss primarily affecting the mid-frequencies.

Procollagen N-proteinase and procollagen C-proteinase. Two unusual metalloproteinases that are essential for procollagen processing probably have important roles in development and cell signaling.

As soon as procollagen precursors of fibrillar collagens were discovered in the early 1970s, it became apparent that connective tissues must contain proteolytic activities that cleave the N-propeptides and the C-propeptides from procollagens. Isolation and characterization of the enzymic activities, however, proved to be unexpectedly difficult. Both proteinases are large and are synthesized in several different forms with polypeptide chains ranging in size from 70 kDa to about 130 kDa. The N-proteinase has the unusual property of cleaving the N-propeptides from type I and type II procollagens if the proteins are in a native conformation, but not if the proteins are partially unfolded so that the N-telopeptides are no longer in a hair-pin configuration. The C-proteinase specifically cleaves native and denatured types I, II and III procollagens. It also specifically cleaves a precursor of lysyl oxidase and laminin 5. Both enzymes and their variants have structures that place them in a large and expanding super-family of over 200 zinc-binding metalloproteinases. The larger of two forms of the N-proteinase contains an RGD sequence for binding through integrins and properdin repeats similar to those found in thrombospondin. The shorter 70 kDa form of the C-proteinase is identical to the protein that was previously identified as bone morphogenic protein-1. Both the 70 kDa C-proteinase and two larger forms are homologous to proteins that are expressed early in development in a variety of organisms, including Drosophila, sea urchin, and fish. Therefore, the data suggest that both the N- and C-proteinases have important biological functions in addition to the roles in the processing of procollagens.

Premature vertebral endplate ossification and mild disc degeneration in mice after inactivation of one allele belonging to the Col2a1 gene for Type II collagen.

Study Design. Skeletal tissues of mice with an inactivated allele of the Col2a1 gene for Type II collagen (“heterozygous knockout”) were studied. Objective. To determine whether a heterozygous inactivation of the Col2a1 gene has a role in the etiology of spine disorders such as disc degeneration. Summary of Background Data. Mutations in the COL2A1, COL11A1, COL11A2, and COL9A2 genes have been linked to spine disorders. However, the mechanism by which genetic factors lead to disc degeneration still are largely unknown. Methods. Spine tissues were studied using radiograph analyses; conventional, quantitative, and polarized light microscopy; immunohistochemistry for the major extracellular components, and in situ hybridization for procollagens &agr;1(I) and &agr;1(II). Voluntary running activity also was monitored in half of the mice. Results. As the findings showed, 1-month-old heterozygous knockout mice had shorter limb bones, skulls, and spines, as well as thicker and more irregular vertebral endplates, which calcified earlier than in the control mice. They also had a lower concentration of glycosaminoglycans in the anulus fibrosus, in the endplates, and in the vertebral bone than the controls. These features in the heterozygous knockout mice were compensated by the age of 15 months. However, the long bones and skulls of the mature heterozygous mice remained shorter than those of the controls. Gene-deficient mice used the running wheel less. However, physical exercise did not induce any marked structural changes in the skeleton. Conclusion. Mice with heterozygous knockout of Col2a1 show subtle early skeletal manifestations that bear some resemblance to those of human spine disorders.

Inactivation of one allele of the type II collagen gene alters the collagen network in murine articular cartilage and makes cartilage softer

OBJECTIVE To evaluate the influence of inactivation of one allele (“heterozygous knockout” or “heterozygous inactivation”) of the type II procollagen gene (Col2a1) on the biomechanical properties and structure of the articular cartilage and subchondral bone in 15 month old mice. METHODS Indentation stiffness of the humerus head articular cartilage was measured by a microindentation method. Cartilage and subchondral bone were prepared for digital densitometry of proteoglycans (PGs), polarised light microscopy (PLM) of collagen, and osteoarthrosis (OA) grading. RESULTS Heterozygous inactivation of the Col2a1 gene softened articular cartilage (p=0.002) as measured by indentation stiffness ((mean (SEM) 0.50 (0.07) MPav 0.94 (0.13) MPa in controls). Fibrillar collagen network exhibited lower birefringence in the intermediate (p=0.04) and deep zones (p=0.01) of cartilage by PLM, indicating either decreased collagen content or a lower degree of fibril parallelism in the knockout mice. The total and zonal thicknesses of articular cartilage were unchanged. Zonal PG contents did not differ significantly. In knockout mice, the prevalence of superficial fibrillation—that is, a sign of OA, was higher than in controls (73%v 21%, p=0.002). The collagen induced birefringence of the superficial zone was not reduced. The subchondral bone volume fraction was lower in knockout mice than in controls, 31%v 43% (p=0.01), and optical retardation values in PLM of bone collagen were slightly but significantly lower (p=0.01). CONCLUSION Heterozygous inactivation of the Col2a1 gene made articular cartilage softer, altered the collagenous network, reduced subchondral bone volume, and altered its microstructure. Changes in the cartilage collagen network probably contributed to increased susceptibility to OA.

Targeted disruption of Col11a2 produces a mild cartilage phenotype in transgenic mice: comparison with the human disorder otospondylomegaepiphyseal dysplasia (OSMED).

Transgenic mice were prepared by homologous recombination with a Col11a2 targeting gene in which an inverted neomycin‐resistant gene was inserted between restriction sites in exons 27 and 28. The targeted allele was transcribed in shortened mRNAs, which could be detected by Northern blotting. However, translation of the full‐length Col11a2 chain was unable to occur because of the presence of premature termination codons within the inverted neomycin‐resistant gene. Analysis of pepsin‐resistant collagen chains from rib cartilage of homozygous mice demonstrated the lack of synthesis of intact α2(XI) chains. However, pepsin‐resistant collagen chains of either α1(XI) or α1(V) were still detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Therefore, α2(XI) chains are not essential for the assembly of some molecular forms of triple‐helical type V/XI collagen. The phenotype was milder than in the cho/cho mouse in which, as the result of mutation, translation of the full‐length α1(XI) chain fails to occur and the mice die at birth (Li et al., 1995 ). Homozygous mice without expression of an α2(XI) chain had a smaller body size, receding snouts, and deafness. Nasal bones in the homozygous transgenic mice were specifically shorter and dimpled on their external surfaces. Chondrocytes in growth plates of all long bones were markedly disorganized and failed to align in columns. Analysis of growth plates from transgenic mice by in situ hybridization showed expression of α1(II) and α1(XI) but not of α1(I) or α1(V) which, in contrast, were expressed in the developing bone and in the bone collar. Expression of α1(X) specifically in the hypertrophic cartilage was observed in normal and transgenic mice. No obvious osteoarthritis was observed throughout the life of homozygous mice up to 1 year of age, although minor morphologic anomalies in the articular cartilages were discernible. The mild phenotype is consistent with similar mutations in the COL11A2 gene seen in patients with nonocular Stickler syndrome and some patients with otospondylomegaepiphyseal dysplasia (OSMED), as well as in patients with a nonsyndromic form of deafness called DFNA13.

The complete cDNA coding sequence for the mouse proα1(I) chain of type I procollagen

The complete sequence of the cDNA for the pro alpha 1 (I) chain of mouse type I procollagen is presented. The encoded amino acid sequence shows 96% identity to the human pro alpha 1 (I) collagen chain.

Recombinant c-proteinase and processes, methods and uses thereof

The present invention is directed to the isolation and identification of the nucleic acid sequence encoding C-proteinase, the recognition of such proteins activity and applications, and tools, processes, and methods of use thereof.

Abnormal response to physical activity in femurs after heterozygous inactivation of one allele of the Col2a1 gene for type II collagen in mice.

The objective of this study was to evaluate the influence of heterozygous inactivation of one allele of the type II collagen gene (Col2a1) on biomechanical properties and mineral density of bone under physical loading conditions. C57BL/6−TGN mice with heterozygous knockout (HZK) inactivation of Col2a1 gene and their nontransgenic littermate controls were housed in individual cages with running wheels for 9 and 15 months. The running activity of each mouse was monitored continuously throughout the experiment. Bone mineral density (BMD) of mice femora was measured using dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computerized tomography (pQCT). Biomechanical properties were determined using three-point bending tests. Vertebral bone samples were prepared for quantitative polarized light microscopy and digital densitometry of proteoglycans. The concentration of total collagen and collagen cross-links were analyzed using high-performance liquid chromatograpy (HPLC).The average daily running distance was shorter for the HZK mice between the age of 4 and 15 months as compared with normal runners (P < 0.05). The ultimate breaking force was 14.8% and 23.6% (9 vs. 15 months) lower in HZK-runners than in wild-type runners. BMD of the femur was 6.1% lower in HZK-runners at the age of 9 months (P < 0.05). Physical activity increased cortical BMD in wild-type runners but not in the HZK runners at the age of 9 months. The collagen network of the HZK mice was less organized. There were only minor changes in BMD and mechanical and structural properties between sedentary HZK mice and their wild-type controls. Increased physical activity induced significantly lower bone density, mechanical properties, and organization of collagen fibers in male HZK mice. However, there were no major differences in biomechanical parameters between sedentary HZK and wild-type male mice. This suggests an important guiding role of collagen type II in bone remodelling and maturation.

cDNA cloning and expression of bovine procollagen I N-proteinase. A new member of the superfamily of zinc-metalloproteinases with binding sites for cells and other matrix components

Procollagen N-proteinase (EC 3.4.24.14) cleaves the amino-propeptides in the processing of type I and type II procollagens to collagens. Deficiencies of the enzyme cause dermatosparaxis in cattle and sheep, and they cause type VIIC Ehlers-Danlos syndrome in humans, heritable disorders characterized by accumulation of pNcollagen and severe skin fragility. Amino acid sequences for the N-proteinase were used to obtain cDNAs from bovine skin. Three overlapping cDNAs had an ORF coding for a protein of 1205 residues. Mammalian cells stably transfected with a complete cDNA secreted an active recombinant enzyme that specifically cleaved type I procollagen. The protein contained zinc-binding sequences of the clan MB of metallopeptidases that includes procollagen C-proteinase/BMP-1. The protein also contained four repeats that are homologous to domains found in thrombospondins and in properdin and that can participate in complex intermolecular interactions such as activation of latent forms of transforming growth factor beta or the binding to sulfatides. Therefore, the enzyme may play a role in development that is independent of its role in collagen biosynthesis. This hypothesis was supported by the observation that in some tissues the levels of mRNA for the enzyme are disproportionately high relative to the apparent rate of collagen biosynthesis.