Te-Cheng Pan

Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells.

The whole amino acid sequence of nidogen was deduced from cDNA clones isolated from expression libraries and confirmed to approximately 50% by Edman degradation of peptides. The protein consists of some 1217 amino acid residues and a 28‐residue signal peptide. The data support a previously proposed dumb‐bell model of nidogen by demonstrating a large N‐terminal globular domain (641 residues), five EGF‐like repeats constituting the rod‐like domain (248 residues) and a smaller C‐terminal globule (328 residues). Two more EGF‐like repeats interrupt the N‐terminal and terminate the C‐terminal sequences. Weak sequence homologies (25%) were detected between some regions of nidogen, the LDL receptor, thyroglobulin and the EGF precursor. Nidogen contains two consensus sequences for tyrosine sulfation and for asparagine beta‐hydroxylation, two N‐linked carbohydrate acceptor sites and, within one of the EGF‐like repeats an Arg‐Gly‐Asp sequence. The latter was shown to be functional in cell attachment to nidogen. Binding sites for laminin and collagen IV are present on the C‐terminal globule but not yet precisely localized.

Mosaic structure of globular domains in the human type VI collagen alpha 3 chain: similarity to von Willebrand factor, fibronectin, actin, salivary proteins and aprotinin type protease inhibitors.

Human collagen alpha 3(VI) chain mRNA (approximately 10 kb) was cloned and shown by sequence analysis to encode a 25 residue signal peptide, a large N‐terminal globule (1804 residues), a central triple helical segment (336 residues) and a C‐terminal globule (803 residues). Some of the sequence was confirmed by Edman degradation of peptides. The N‐terminal globular segment consists of nine consecutive 200 residue repeats (N1 to N9) showing internal homology and also significant identity (17‐25%) to the A domains of von Willebrand Factor and similar domains present in some other proteins. Deletions were found in the N3 and N9 domains of several cDNA clones suggesting variation of these structures by alternative splicing. The C‐terminal globule starts immediately after the triple helical segment with two domains C1 (184 residues) and C2 (248 residues) being similar to the N domains. They are followed by a proline rich, repetitive segment C3 of 122 residues, with similarity to some salivary proteins, and domain C4 (89 residues), which is similar to the type III repeats present in fibronectin and tenascin. The most C‐terminal domain C5 (70 residues) shows 40‐50% identity to a variety of serine protease inhibitors of the Kunitz type. The whole sequence contains 29 cysteines which are mainly clustered in short segments connecting domains N1, C1, C2 and the triple helix, and in the inhibitor domain. Five putative Arg‐Gly‐Asp cell‐binding sequences are exclusively localized in the triple helical segment.(ABSTRACT TRUNCATED AT 250 WORDS)

New molecular mechanism for Ullrich congenital muscular dystrophy: a heterozygous in-frame deletion in the COL6A1 gene causes a severe phenotype.

Recessive mutations in two of the three collagen VI genes, COL6A2 and COL6A3, have recently been shown to cause Ullrich congenital muscular dystrophy (UCMD), a frequently severe disorder characterized by congenital muscle weakness with joint contractures and coexisting distal joint hyperlaxity. Dominant mutations in all three collagen VI genes had previously been associated with the considerably milder Bethlem myopathy. Here we report that a de novo heterozygous deletion of the COL6A1 gene can also result in a severe phenotype of classical UCMD precluding ambulation. The internal gene deletion occurs near a minisatellite DNA sequence in intron 8 that removes 1.1 kb of genomic DNA encompassing exons 9 and 10. The resulting mutant chain contains a 33-amino acid deletion near the amino-terminus of the triple-helical domain but preserves a unique cysteine in the triple-helical domain important for dimer formation prior to secretion. Thus, dimer formation and secretion of abnormal tetramers can occur and exert a strong dominant negative effect on microfibrillar assembly, leading to a loss of normal localization of collagen VI in the basement membrane surrounding muscle fibers. Consistent with this mechanism was our analysis of a patient with a much milder phenotype, in whom we identified a previously described Bethlem myopathy heterozygous in-frame deletion of 18 amino acids somewhat downstream in the triple-helical domain, a result of exon 14 skipping in the COL6A1 gene. This deletion removes the crucial cysteine, so that dimer formation cannot occur and the abnormal molecule is not secreted, preventing the strong dominant negative effect. Our studies provide a biochemical insight into genotype-phenotype correlations in this group of disorders and establish that UCMD can be caused by dominantly acting mutations.

The role of the alpha3(VI) chain in collagen VI assembly. Expression of an alpha3(VI) chain lacking N-terminal modules N10-N7 restores collagen VI assembly, secretion, and matrix deposition in an alpha3(VI)-deficient cell line.

Collagen VI is a microfibrillar protein found in the extracellular matrix of virtually all connective tissues. Three genetically distinct subunits, the α1(VI), α2(VI), and α3(VI) chains, associate intracellularly to form triple-helical monomers, which then assemble into disulfide-bonded dimers and tetramers before secretion. Although sequence considerations suggest that collagen VI monomers composed of all three chains are the most stable isoform, the precise chain composition of collagen VI remains controversial and alternative assemblies containing only α1(VI) and α2(VI) chains have also been proposed. To address this question directly and study the role of the α3(VI) chain in assembly, we have characterized collagen VI biosynthesis and in vitro matrix formation by a human osteosarcoma cell line (SaOS-2) that is deficient in α3(VI) production. Northern analysis showed an abundance of α1(VI) and α2(VI) mRNAs, but no detectable α3(VI) mRNA was apparent in SaOS-2 cells. By day 30 of culture, however, small amounts of α3(VI) mRNA were detected, although the level of expression was still much less than α1(VI) and α2(VI). Collagen VI protein was not detected in SaOS-2 medium or cell layer samples until day 30 of culture, demonstrating that despite the abundant synthesis of α1(VI) and α2(VI), no stable collagen VI protein was produced without expression of α3(VI). The α1(VI) and α2(VI) chains produced in the absence of α3(VI) were non-helical and were largely retained intracellularly and degraded. The critical role of the α3(VI) chain in collagen VI assembly was directly demonstrated after stable transfection of SaOS-2 cells with an α3(VI) cDNA expression construct that lacked 4 of the 10 N-terminal type A subdomains. The transfected α3(VI) N6–C5 chains associated with endogenous α1(VI) and α2(VI) and formed collagen VI dimers and tetramers, which were secreted and deposited into an extensive network in the extracellular matrix. These data demonstrated that α3(VI) is essential for the formation of stable collagen VI molecules and subdomains N10–N7 are not required for molecular assembly.

Fibulin-2 Is Dispensable for Mouse Development and Elastic Fiber Formation

ABSTRACT Fibulin-2 is an extracellular matrix protein belonging to the five-member fibulin family, of which two members have been shown to play essential roles in elastic fiber formation during development. Fibulin-2 interacts with two major constituents of elastic fibers, tropoelastin and fibrillin-1, in vitro and localizes to elastic fibers in many tissues in vivo. The protein is prominently expressed during morphogenesis of the heart and aortic arch vessels and at early stages of cartilage development. To examine its role in vivo, we generated mice that do not express the fibulin-2 gene (Fbln2) through homologous recombination of embryonic stem cells. Unexpectedly, the fibulin-2-null mice were viable and fertile and did not display gross and anatomical abnormalities. Histological and ultrastructural analyses revealed that elastic fibers assembled normally in the absence of fibulin-2. No compensatory up-regulation of mRNAs for other fibulin members was detected in the aorta and skin tissue. However, in the fibulin-2 null aortae, fibulin-1 immunostaining was increased in the inner elastic lamina, where fibulin-2 preferentially localizes. The results demonstrate that fibulin-2 is not required for mouse development and elastic fiber formation and suggest possible functional redundancy between fibulin-1 and fibulin-2.

COL6A1 genomic deletions in Bethlem myopathy and Ullrich muscular dystrophy

We have identified highly similar heterozygous COL6A1 genomic deletions, spanning from intron 8 to exon 13 or intron 13, in two patients with Ullrich congenital muscular dystrophy and the milder Bethlem myopathy. The 5′ breakpoints of both deletions are located within a minisatellite in intron 8. The mutations cause in‐frame deletions of 66 and 84 amino acids in the amino terminus of the triple‐helical domain, leading to intracellular accumulation of mutant polypeptides and reduced extracellular collagen VI microfibrils. Our studies identify a deletion‐prone region in COL6A1 and suggest that similar mutations can lead to congenital muscle disorders of different clinical severity. Ann Neurol 2005

Tumor-specific expression and alternative splicing of the COL6A3 gene in pancreatic cancer

BACKGROUND Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease; a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagens III and V, comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked with inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events. METHODS We analyzed paired PDA-normal (n = 18), intraductal papillary mucinous neoplasms (IPMN; n = 5), pancreatic cystadenoma (n = 5), and 8 PDA cell lines with reverse transcriptase polymerase chain reaction, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in 2 animal models of PDA. RESULTS COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA-adjacent tissues. Inclusion of exon 4 was exclusively tumor specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in 6 PDA cell lines, but only 2 cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma and some PDA cells. CONCLUSION We have described, for the first time, a dynamic process of tumor-specific alternative splicing in several exons of stromal COL6A3. Alternatively spliced proteins may contribute to the etiology or progression of cancer and may serve as markers for cancer diagnosis. Identification of COL6A3 isoforms as PDA-specific provides the basis for future studies to explore the oncogenic and diagnostic potential of these alternative splicing events.

The exon organization of the triple-helical coding regions of the human α1(VI) and α2(VI) collagen genes is highly similar

Abstract The α1(VI) and α2(VI) chains, two of the three constituent chains of type VI collagen, are highly similar in size and domain structure. They are encoded by single-copy genes residing in close proximity on human chromosome 21. To study the evolution of the type VI collagen genes, we have isolated and characterized genomic clones coding for the triple-helical domains of the human α1(VI) and α2(VI) chains, which consist of 336 and 335 amino acid residues, respectively. Nucleotide sequencing indicates that, in both genes, the exons are multiples of 9 bp in length (including 27, 36, 45, 54, 63, and 90 bp) except for those encoding for regions with triple-helical interruptions. In addition, the introns are positioned between complete codons. The most predominant exon size is 63 bp, instead of 54 bp as seen in the fibrillar collagen genes. Of particular interest is the finding that the exon structures of the α1(VI) and α2(VI) genes are almost identical. A significant deviation is that a segment of 30 amino acid residues is encoded by two exons of 54 and 36 bp in the α1(VI) gene, but by a single exon of 90 bp in the α2(VI) gene. The exon arrangement therefore provides further evidence that the two genes have evolved from tandem gene duplication. Furthermore, comparison with the previously reported gene structure of the chick α2(VI) chain indicates that the exon structure for the triple-helical domain of the α2(VI) collagen is strictly conserved between human and chicken.

A homozygous COL6A2 intron mutation causes in-frame triple-helical deletion and nonsense-mediated mRNA decay in a patient with Ullrich congenital muscular dystrophy

Ullrich congenital muscular dystrophy (UCMD) is a severe disorder caused, in most cases, by a deficiency in collagen VI microfibrils. Recessive mutations in two of the three collagen VI genes, COL6A2 and COL6A3, have been identified in eight of the nine UCMD patients reported thus far. A heterozygous COL6A1 gene deletion, resulting in a mutant protein that exerts a dominant negative effect, has recently been described in a severely affected UCMD patient. Here we describe a patient in whom reverse transcription-PCR analysis of fibroblast RNA suggested a heterozygous in-frame deletion of exon 13 in the triple-helical domain of COL6A2, which is predicted to be dominantly acting. However, a homozygous A→G mutation at −10 of intron 12 was found in the genomic DNA. The intron mutation activated numerous cryptic splice acceptor sites, generating normal and exon 13-deleted COL6A2 mRNA, and multiple aberrant transcripts containing frameshifts that were degraded through a nonsense-mediated decay mechanism. Northern analysis indicated diminished COL6A2 mRNA expression as the primary pathogenic mechanism in this UCMD patient. Our results underscore the importance of multifaceted analyses in the accurate molecular diagnosis and interpretation of genotype-phenotype correlations of UCMD.

Zinc Finger Protein Zac1 Is Expressed in Chondrogenic Sites of the Mouse

Zac1 is a zinc finger transcription factor that elicits antiproliferative activity and is a potential tumor suppressor gene. Through a detailed spatiotemporal study by in situ hybridization of mouse embryos, we have found that Zac1 transcript is predominantly localized in developing chondrogenic tissue, in addition to the central nervous system as reported elsewhere. Zac1 is also expressed transiently in the myocardium, skeletal muscle, and basal aspect of the stratified embryonic epithelia. During cartilage development, the pattern of Zac1 expression is in close accordance with the distribution of type II collagen mRNA in mesenchymal condensation and prehypertrophic chondrocytes. In mouse ATDC5 cells undergoing in vitro chondrogenesis, the Zac1 mRNA is up‐regulated in parallel with genes expressed in precartilage but the Zac1 expression is low when type II collagen mRNA is markedly increased in differentiated cells. Together, these results suggest that Zac1 is a potential regulatory gene involved in chondrogenic differentiation. Developmental Dynamics 229:340–348, 2004.