Intracellular membranes in the synthesis, transport, and metabolism of proteoglycans

150

127

We recently cloned human chondroitin synthase (ChSy) exhibiting the glucuronyltransferase-II (GlcAT-II) and N-acetylgalactosaminyltransferase-II (GalNAcT-II) activities responsible for the biosynthesis of repeating disaccharide units of chondroitin sulfate, but chondroitin polymerization was not demonstrated in vitro using the recombinant ChSy. We report here that the chondroitin polymerizing activity requires concomitant expression of a novel protein designated chondroitin polymerizing factor (ChPF) with ChSy. The human ChPF consists of 775 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 23% identity to that of human ChSy. The expression of a soluble recombinant form of the protein in COS-1 cells produced a protein with little GlcAT-II or GalNAcT-II activity. In contrast, coexpression of the ChPF and ChSy yielded markedly augmented glycosyltransferase activities, whereas simple mixing of the two separately expressed proteins did not. Moreover, using both UDP-glucuronic acid (GlcUA) and UDP-N-acetylgalactosamine (GalNAc) as sugar donors, chondroitin polymerization was demonstrated on the so-called glycosaminoglycan-protein linkage region tetrasaccharide sequence of ␣-thrombomodulin. These results suggested that the ChPF acts as a specific activating factor for ChSy in chondroitin polymerization. The coding region of the ChPF was divided into four discrete exons and localized to chromosome 2q35-q36. Northern blot analysis revealed that the ChPF gene exhibited a markedly different expression pattern among various human tissues, which was similar to that of ChSy. Thus, the ChPF is required for chondroitin polymerizing activity of mammalian ChSy.

Section: Discussioncontrasting

confidence: 67%

Section: Discussioncontrasting

confidence: 48%

Molecular Cloning of a Chondroitin Polymerizing Factor That Cooperates with Chondroitin Synthase for Chondroitin Polymerization

Kitagawa

Izumikawa

Uyama

et al. 2003

150

127

“…In some core proteins, potential Ser-Gly xylosylation sites often escape xylosylation (43). We compared different proteoglycans and found that those with chondroitin sulfate chains of substantial importance for the function of the protein, e.g.…”

Section: Sequence Homology Of the Glycosylation Sites Of Differentmentioning

confidence: 99%

Recognition of Acceptor Proteins by UDP-D-xylose Proteoglycan Core Protein β-D-Xylosyltransferase

Brinkmann

Weilke

Kleesiek

1997

The formation of chondroitin sulfate is initiated by xylosyltransferase (XT) transferring xylose from UDPxylose to consensus serine residues of proteoglycan core proteins. Our alignment of 51 amino acid sequences of chondroitin sulfate attachment sites in 19 different proteins resulted in a consensus sequence for the recognition signal of XT. The complete recognition sequence is composed of the amino acids a-a-a-a-G-S-G-a-b-a, with a ‫؍‬ E or D and b ‫؍‬ G, E, or D. This sequence was confirmed by determination of the Michaelis-Menten constants for in vitro xylosylation of different synthetic proteins and peptides using an enriched XT preparation from conditioned cell culture supernatant of human chondrocytes. The highest acceptor activity was determined by the sequence Q-E-E-E-E-G-S-G-G-G-Q, which was found in the single chondroitin sulfate attachment site of bikunin, the inhibitory active component of the human inter-␣-trypsin inhibitor.We determined the Michaelis-Menten constant (K m ) of xylosylation of the synthetic bikunin analogous peptide Q-E-E-E-G-S-G-G-G-Q-K to be 22 M, which was 9-fold decreased in comparison to deglycosylated core protein from bovine cartilage (188 M), which was previously used as acceptor for the XT activity assay. The best XT acceptors were nonglycosylated recombinant wildtype bikunin (K m ‫؍‬ 0. These results imply that the primary structure of the acceptor is not the only determinant for recognition by xylosyltransferase. Thus, protein conformation is also a main factor in determining xylosylation.

“…The average molecular weight of oligosaccharide number 5 was 2176.5, which corresponded to an octasaccharide with eight sulfated residues, (HexA) 4 (HexNA) 4 (OSO 3 H) 8 . HPLC analysis of the chondroitinase AC-II digest of number 5 after derivatization with PA showed PA derivatives of Di-diS E and ⌬Di-diS E in a molar ratio of 1.0:3.0 (Table II).…”

Section: Figmentioning

confidence: 99%

Domain Structure of Chondroitin Sulfate E Octasaccharides Binding to Type V Collagen

Takagaki

Munakata

Kakizaki

et al. 2002

We demonstrated previously that chondroitin sulfate E (ChS-E) binds to type V collagen (Munakata, H., Takagaki, K., Majima, M., and Endo, M. (1999) Glycobiology 9, 1023-1027). In this study, we investigated the structure and binding of ChS-E oligosaccharides. Eleven oligosaccharides were isolated from ChS-E by gel filtration chromatography and anion-exchange high performance liquid chromatography after hydrolysis with testicular hyaluronidase. Separately, seven oligosaccharides were custom synthesized using the transglycosylation reaction of testicular hyaluronidase. Structural analysis was performed by enzymatic digestions in conjunction with high performance liquid chromatography and mass spectrometry. This library of 18 oligosaccharides was used as a source of model molecules to clarify the structural requirements for binding to type V collagen. Binding was analyzed by a biosensor based on surface plasmon resonance. The results indicated that to bind to type V collagen the oligosaccharides must have the following carbohydrate structures: 1) octasaccharide or larger in size; 2) a continuous sequence of three GlcA␤1-3GalNAc(4S,6S) units; 3) a GlcA␤1-3GalNAc(4S,6S) unit, GlcA␤1-3GalNAc(4S) unit or GlcA␤1-3GalNAc(6S) unit at the reducing terminal; 4) a GlcA␤1-3GalNAc(4S,6S) unit at the nonreducing terminal. It is likely that these characteristic oligosaccharide sequences play key roles in cell adhesion and extracellular matrix assembly.Proteoglycan exists in tissues of many mammalian species and is widely distributed in the cell surface and extracellular matrix. It is known that its carbohydrate chain, chondroitin sulfate (ChS), 1 has various important biological activities in areas such as cell migration, recognition, and morphogenesis (1-5). Recently, greater attention has been directed toward the functions of ChS in the brain, optic nerves, and chondrocytes (6 -8). ChS has many types of structural domains that are known to participate in specific physiological functions. However, the relationship between the biological function and structure of ChS domains is not yet fully understood. Recently, we used a surface plasmon resonance (SPR) biosensor to investigate the interaction of glycosaminoglycans (GAGs) with collagens and glycoproteins from the extracellular matrix (9). It was found that chondroitin sulfate E (ChS-E) has specific affinity for type V collagen, and that a novel characteristic sequence included in ChS-E is probably involved in binding to type V collagen. ChS-E from squid cartilage consists mainly of the disaccharide units GlcA␤1-3GalNAc(4S,6S), GlcA␤1-3GalNAc(4S), GlcA␤1-3GalNAc(6S), and GlcA␤1-3GalNAc, where 4S and 6S represent 4-O-and 6-O-sulfate, respectively (10, 11). Thus, since ChS-E has a variety of structures, it is likely that some specific feature of its structure is necessary for binding to type V collagen. However, the structural features within ChS-E that bind to type V collagen remain unclear.In the present work, we have prepared various oligosaccharides by testicular hyaluronidase...