Synthesis of Proteoglycan Using the Endo-type Glycosidase

Endo-type glycosidases were investigated with the aim of performing glycotechnological synthesis of artificial proteoglycan (PG).

Fig.1 One Example of Custom-synthesized Glycosaminoglycan Sugar Chain

1. Synthesis of the glycosaminoglycan (GAG) sugar chain using enzymatic reconstruction.

The GAG sugar chains are high molecule polysaccharides (molecular weight: roughly 10,000 ~ 2 million) constructed with a number of disaccharide repeating units consisting of uronic acid (-glucuronic acid or -iduronic acid) and hexosamine (N-acetylgalactosamine or N-acetylglucosamine). They are divided roughly into hyaluronic acid (HA), chondroitin sulfate (ChS), dermatan sulfate (DS), heparan sulfate (HS), heparin (Hep), and keratan sulfate (galactose containing instead of uronic acid). However, because the inside of GAG sugar chains are not only uniform disaccharide repeating units but show diversity in number and position of the sulfate groups, GAGs have various physiological active domains.

Endo-type glycosidase acting on the GAG sugar chain is the testicular hyaluronidase (endo--N-acetylhexosaminidase), and this enzyme acts on not only HA but also chondroitin (Ch), chondroitin 4-sulfate (Ch4S) and chondroitin 6-sulfate (Ch6S). Besides the hydrolytic reaction, hyaluronidase is also known to catalyze the reverse reaction, transglycosylation. This reconstruction of the sugar chain occurs when the transglycosylation reaction occurs between different types of GAG as donors or acceptors. Therefore, it is possible to custom-synthesize the GAG sugar chains by controlling their combinations. For example, the synthetic process of decasaccharide with different arrangements of sulfate groups at the non-reducing terminal, GlcA1-3GalNAc(4S)1-4GlcA1-3GalNAc1-4GlcA1-3GalNAc(6S), is described in Figure 1. First, transglycosylation is done using Ch6S hexasaccharide composed of N-acetylgalactosamine with a 6-sulfate group (GalNAc(6S)) as an acceptor and Ch composed of N-acetylgalactosamine without a sulfate group (GalNAc) as a donor. Second, transglycosylation is done using synthesized octasaccharide as the next acceptor and Ch4S composed of N-acetylgalactosamine with a 4-sulfate group (GalNAc(4S)) as a donor. Finally, it is possible to reconstruct new decasaccharides with three types of disaccharide units.

Fig.2 Scheme of the Transglycosylation Reaction by Endo--xylosidase

2. Endo-type glycosidase is useful for transfer of the GAG sugar chain to the protein.

PGs consist of a core protein and covalently attached GAG chains. Although ChS, DS, HS, and Hep are structurally different, every GAG chain of these is covalently attached to the serine residue of core protein through the common linkage region (GlcA-Gal-Gal-Xyl).

It is known that there are three kinds of endo-type glycosidases endo--xylosidase, endo--galactosidase, and endo--glucuronidase; which specifically act on the linkage region of the GAG sugar chain and the core protein of PG. Because endo--xylosidase hydrolyzes the Xyl-Ser linkage, the binding site of the GAG sugar chain and the core protein, GAG sugar chains are especially easy to liberate intact. Therefore, this enzyme is an important tool in the GAG sugar chain analysis of PG. Another important point is the reverse reaction of hydrolysis,the transglycosylation reaction. When the transglycosylation activity using a ChS sugar chain with the peptide as the donor and p-nitrophenyl-glycerol (pNP-glycerol) as the acceptor was analyzed, it was shown that transglycosylation activity transfers ChS sugar in a chains of 40,000 molecular weight to the acceptor (Fig. 2). In addition, it is possible that DS and HS also transfer similar manner. Therefore, future developments to be expected include: restoration by genetic-engineering of PG lacking in the GAG sugar chain, modification of the GAG sugar chain of natural PG, and synthesis of new functional PG added to the artificial GAG sugar chain by enzymatic reconstruction.

Keichi Takagaki:(School of Medicine,Hirosaki University)
References (1) Takagaki K, Kon A, Kawasaki H, Nakamura T, Tamura S, and Endo M: Isolation and characterization of Patinopecten mid-gud gland endo--xylosidase active on peptidochondroitin sulfate. J. Biol. Chem.265, 854-860, 1990
(2) Takagaki K, Ishido K: Synthesis of chondroitin sulfate oligosaccharides using enzymatic reconstruction. Trends. Glycosci. Glyc. 12, 295-306, 2000
Dec.15, 2001

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