Enzymatic Synthesis of Chondroitin with a Novel Chondroitin Sulfate N-Acetylgalactosaminyltransferase That Transfers N-Acetylgalactosamine to Glucuronic Acid in Initiation and Elongation of Chondroitin Sulfate Synthesis

Abstract

We found a novel glycosyltransferase gene having a hypothetical ␤1,4-galactosyltransferase motif (GenBank TM accession number AB081516) by a BLAST search and cloned its full-length open reading frame using the 5-rapid amplification of cDNA ends method. The truncated form was expressed in insect cells as a soluble enzyme. It transferred N-acetylgalactosamine, not galactose, to para-nitrophenyl-␤-glucuronic acid. The N-acetylgalactosamine-glucuronic acid linkage has been identified only in chondroitin sulfate; therefore, we examined its chondroitin elongation and initiation activities. N-Acetylgalactosaminyltransferase activity was observed toward chondroitin poly-and oligosaccharides, chondroitin sulfate oligosaccharides, and linkage tetrasaccharide (GlcA-Gal-Gal-Xyl-O-methoxyphenyl), and the chondroitin polysaccharide and linkage tetrasaccharide were better acceptor substrates than the others. Northern blot analysis and quantitative realtime PCR analysis revealed that its 4-kb transcripts were highly expressed in thyroid and placenta, although they were ubiquitously expressed in various tissues and cells. These results suggest that this enzyme has N-acetylgalactosaminyltransferase activity in both the elongation and initiation of chondroitin sulfate synthesis. Furthermore, we performed enzymatic synthesis of chondroitin pentasaccharide in vitro. In one tube reaction with four enzymes, ␤1,4-galactosyltransferase-VII, ␤1,3-galactosyltransferase-VI, glucuronyltransferase-I, and this enzyme, and a synthetic xylose-peptide acceptor, the structure GalNAc-GlcA-Gal-Gal-Xyl-peptide was constructed. This is the first report of a chondroitin pentasaccharide constructed with recombinant glycosyltransferases in vitro.

Chondroitin Sulfate N-Acetylgalactosaminyltransferase-1 Plays a Critical Role in Chondroitin Sulfate Synthesis in Cartilage

“…In tissues other than cartilage, CSGalNacT-1 is expressed at the highest level in the thyroid gland and placenta, which exhibit expression only up to 1.5-5-fold that of CSGalNAcT-2 (20). In addition, various cell lines show relatively low levels of CSGalNAcT-1 expression compared with other enzymes (18). 3 Thus, the high expression of CSGalNAcT-1 appears to be characteristic of chondrocytes and car-3 K. Sakai, unpublished data.…”

“…We designate this molecule "superaggrecan," since it would more efficiently contribute to cartilage function. Since CSGalNAcT-1 has stronger initiating activity than elongating activity (18), chain initiation may be the rate-limiting step in CS biosynthesis, and chondrocytes may have sufficient machinery for CS chain elongation following initiation. Indeed, overexpression of CSS-1, CSS-2, and CSGlcAT did not increase CS biosynthesis.…”

“…To date, six glycosyltransferases involved in CS synthesis have been identified (Fig. 1B): chondroitin sulfate synthase-1 (CSS-1)/chondroitin synthase (13), chondroitin sulfate synthase-2 (CSS-2)/chondroitin-polymerizing factor (14,15), chondroitin sulfate synthase-3 (CSS-3) (16), chondroitin sulfate glucuronyltransferase (CSGlcAT) (17), and chondroitin sulfate N-acetylgalactosaminyltransferase-1 (18,19) and -2 (20, 21) (CSGalNAcT-1 and -2, respectively). All of these enzymes have an N-terminal transmembrane domain and are localized to the Golgi apparatus, where CS biosyn-thesis takes place (22).…”

Molecular Cloning and Expression of a Second Chondroitin N-Acetylgalactosaminyltransferase Involved in the Initiation and Elongation of Chondroitin/Dermatan Sulfate

“…The first chondroitin glycosyltransferase cloned was chondroitin synthase consisting of a single large polypeptide with dual glycosyltransferase activities of GlcUA transferase II (Glc-AT-II) and GalNAc transferase II (GalNAcT-II) that is responsible for synthesizing the repeating disaccharide units of chondroitin sulfate (18). Chondroitin GalNAcT-1, the second chondroitin glycosyltransferase cloned, exhibits GalNAcT-II activity for chain elongation and GalNAc transferase I (GalNAcT-I) activity that determines and initiates the synthesis of chondroitin sulfate on the common GAG-protein linkage region (19,20,22). Chondroitin GlcUA transferase, the third chondroitin glycosyltransferase cloned, has only GlcAT-II activity, which has been proposed to be involved in chain elongation (21).…”

Molecular Cloning and Characterization of a Novel Human β1,4-N-Acetylgalactosaminyltransferase, β4GalNAc-T3, Responsible for the Synthesis of N,N′-Diacetyllactosediamine, GalNAcβ1–4GlcNAc

“…In the case of CSSs, the ␤4GalNAc-T domain exists in the C-terminal region. Chondroitin sulfate Nacetylgalactosaminyltransferase I and II (CSGalNAc-T1 and -T2) both act as ␤4GalNAc-Ts in the initiation and elongation of chondroitin sulfate synthesis (13)(14)(15)(16).…”