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Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher Km value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase. [ABSTRACT FROM AUTHOR]

We characterized recombinant Lin1839 protein (Lin1839r) belonging to glycoside hydrolase family 94 from Listeria innocua. Lin1839r catalyzed the synthesis of a series of 1,2-β-oligoglucans (Sopn: n denotes degree of polymerization) using sophorose (Sop2) as the acceptor and α-d-glucose 1-phosphate (Glc1P) as the donor. Lin1839r recognized glucose as a very weak acceptor substrate to form polymeric 1,2-β-glucan. The degree of polymerization of the 1,2-β-glucan gradually decreased with long-term incubation to generate a series of Sopns. Kinetic analysis of the phosphorolytic reaction towards sophorotriose revealed that Lin1839r followed a sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of sophorotetraose and sophoropentaose were similar to those of sophorotriose, although the enzyme did not exhibit significant phosphorolytic activity on Sop2. These results indicate that the Lin1839 protein is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of 1,2-β-glucan with a degree of polymerization of ≥3. We propose 1,2-β-oligoglucan: phosphate α-glucosyltransferase as the systematic name and 1,2-β-oligoglucan phosphorylase as the short name for this Lin1839 protein. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is one of the most prevalent skeletal diseases. Recently, we identified a novel gene on chromosome 3p24.3, named DVWA (double von Willebrand factor A domains), and its functional variants, which are associated with susceptibility to knee OA. Here we report the cloning and characterization of the DVWA gene. DVWA consisted of seven exons and had four alternative splicing variants, which encoded long (385 amino acid) and short (276 amino acid) proteins (L-DVWA and S-DVWA, respectively). S-DVWA was an N-terminal truncated form of L-DVWA and lacked a signal peptide and a part of a VWA domain. L-DVWA and S-DVWA transcripts were mainly expressed in articular cartilage. Immunoblot analysis using epitope-tagged proteins showed L-DVWA in the conditioned media and S-DVWA only in the cell, consistent with the in silico prediction. We also cloned the murine counterpart of DVWA, which was found to be identical to Col6a4, which has recently been reported. L-DVWA had 73% identity to the N-terminal sequence of the 2,309-amino acid Col6a4 protein. The mouse Dvwa/Col6a4 mRNA was present mainly in the small intestine in embryos and adults, but not in cartilage. The amino acid sequence of L-DVWA was conserved in higher species than chicken, but that of S-DVWA was unique in human. Knockdown of DVWA by siRNAs increased expression of chondrocyte matrix genes. Our study indicates that DVWA is evolutionally very unique, which, together with its specific expression in articular cartilage, suggests its specific role in human cartilage metabolism. [ABSTRACT FROM AUTHOR]

Abstract: Cartilage intermediate layer protein (CILP) is an extracellular matrix protein abundant in cartilaginous tissues. CILP is implicated in common musculoskeletal disorders, including osteoarthritis and lumbar disc disease. Regulation of the CILP gene is largely unknown, however. We have found that CILP mRNA expression is induced by TGF-β1 and dependent upon signaling via TGF-β receptors. TGF-β1 induction of CILP is mediated by Smad3, which acts directly through cis-elements in the CILP promoter region. Pathways other than Smad3 also are involved in TGF-β1 induction of CILP. These observations, together with the finding that CILP protein binds and inhibits TGF-β1, suggest that CILP and TGF-β1 may form a functional feedback loop that controls chondrocyte metabolism. [Copyright &y& Elsevier]

Presents a study on transglycossylation activity of Dictyoglomus thermophilum amylase A. Characteristics of amylase A from Dictyoglamus thermophilum; Examination of the substrate specificity and reaction products of amylase A from D. thermophilum; Results that suggest the enzyme was able to use maltooligosaccharides and long chain amylose as substrate and has transglycosylation activity.

Axial spondylometaphyseal dysplasia (axial SMD) is an autosomal recessive disease characterized by dysplasia of axial skeleton and retinal dystrophy. We conducted whole exome sequencing and identified C21orf2 (chromosome 21 open reading frame 2) as a disease gene for axial SMD. C21orf2 mutations have been recently found to cause isolated retinal degeneration and Jeune syndrome. We found a total of five biallelic C21orf2 mutations in six families out of nine: three missense and two splicing mutations in patients with various ethnic backgrounds. The pathogenic effects of the splicing (splice-site and branch-point) mutations were confirmed on RNA level, which showed complex patterns of abnormal splicing. C21orf2 mutations presented with a wide range of skeletal phenotypes, including cupped and flared anterior ends of ribs, lacy ilia and metaphyseal dysplasia of proximal femora. Analysis of patients without C21orf2 mutation indicated genetic heterogeneity of axial SMD. Functional data in chondrocyte suggest C21orf2 is implicated in cartilage differentiation. C21orf2 protein was localized to the connecting cilium of the cone and rod photoreceptors, confirming its significance in retinal function. Our study indicates that axial SMD is a member of a unique group of ciliopathy affecting skeleton and retina. [ABSTRACT FROM AUTHOR]