2137

EXTL3

BOTV|EXTR1|REG|REGR|RPR;exostoses (multiple)-like 3;EXTL3;exostoses (multiple)-like 3

EXT-related protein 1|exostosin-like 3|glucuronyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase|hereditary multiple exostoses gene isolog|multiple exostosis-like protein 3|putative tumor suppressor protein EXTL3|reg receptor

8p21

protein-coding

Count: 2; TAG,Generif

Exostoses like-3 (EXTL3) is a putative tumour suppressor gene but its involvement in colorectal cancer (CRC) is unclear. We have investigated the role of methylation of the EXTL3 promoter as a mechanism for EXTL3 regulation and tested the hypothesis that loss of EXTL3 expression is associated with mucinous differentiation and alteration of glycoprotein expression in CRC cells. The methylation status of the EXTL3 gene promoter was analysed by methylation-specific PCR following bisulphite modification in CRC cell lines and microdissected primary CRC tissues and their corresponding adjacent normal colorectal mucosa. EXTL3 promoter methylation was detected in seven of 11 mucinous CRCs (63.6%) but in none of 26 non-mucinous CRCs examined. EXTL3 promoter methylation was also detected in the normal colonic mucosa of three patients with mucinous CRC but not in the normal colonic mucosa of any patients with non-mucinous CRC. The presence of EXTL3 methylation was significantly associated with the partial loss of HS expression in mucinous CRC lesions. The mucinous CRC cell lines, Colo201 and Colo205, showed EXTL3 promoter methylation and loss of EXTL3 mRNA expression. However 5-aza-2'-deoxycytidine treatment demethylated the EXTL3 gene promoter and restored its mRNA expression. Furthermore, the basal expression of HS in CRC cells was abolished by treatment with EXTL3-siRNA. We conclude that EXTL3 promoter methylation and its related loss of EXTL3 expression are involved in the loss of HS expression in mucinous CRCs.

The tumor suppressors EXT1 and EXT2 are associated with hereditary multiple exostoses and encode bifunctional glycosyltransferases essential for chain polymerization of heparan sulfate (HS) and its analog, heparin (Hep). Three highly homologous EXT-like genes, EXTL1-EXTL3, have been cloned, and EXTL2 is an alpha1,4-GlcNAc transferase I, the key enzyme that initiates the HS/Hep synthesis. In the present study, truncated forms of EXTL1 and EXTL3, lacking the putative NH2-terminal transmembrane and cytoplasmic domains, were transiently expressed in COS-1 cells and found to harbor alpha-GlcNAc transferase activity. EXTL3 used not only N-acetylheparosan oligosaccharides that represent growing HS chains but also GlcAbeta1-3Galbeta1-O-C2H4NH-benzyloxycarbonyl (Cbz), a synthetic substrate for alpha-GlcNAc transferase I that determines and initiates HS/Hep synthesis. In contrast, EXTL1 used only the former acceptor. Neither EXTL1 nor EXTL3 showed any glucuronyltransferase activity as examined with N-acetylheparosan oligosaccharides. Heparitinase I digestion of each transferase-reaction product showed that GlcNAc had been transferred exclusively through an alpha1,4-configuration. Hence, EXTL3 most likely is involved in both chain initiation and elongation, whereas EXTL1 possibly is involved only in the chain elongation of HS and, maybe, Hep as well. Thus, their acceptor specificities of the five family members are overlapping but distinct from each other, except for EXT1 and EXT2 with the same specificity. It now has been clarified that all of the five cloned human EXT gene family proteins harbor glycosyltransferase activities, which probably contribute to the synthesis of HS and Hep.