Human tumor suppressor EXT gene family members EXTL1 and EXTL3 encode α1,4- N -acetylglucosaminyltransferases that likely are involved in heparan sulfate/ heparin biosynthesis

Abstract

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 ␣1,4-GlcNAc transferase I, the key enzyme that initiates the HS͞Hep synthesis. In the present study, truncated forms of EXTL1 and EXTL3, lacking the putative NH 2-terminal transmembrane and cytoplasmic domains, were transiently expressed in COS-1 cells and found to harbor ␣-GlcNAc transferase activity. EXTL3 used not only N-acetylheparosan oligosaccharides that represent growing HS chains but also GlcA␤1-3Gal␤1-O-C 2H4NH-benzyloxycarbonyl (Cbz), a synthetic substrate for ␣-GlcNAc transferase I that determines and initiates HS͞Hep 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 ␣1,4-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.

Heparin and Heparan Sulfate Biosynthesis

“…Identification of EXTL2 as GlcNAcT-I for HS/heparin synthesis suggested that other EXT-like genes also have some roles in HS formation. In fact, we demonstrated that EXTL1 possesses only the GlcNAcT-II activity whereas EXTL3 shows both GlcNAcT-I and -II activities (56). Neither EXTL1 nor EXTL3 show any glucuronyltransferase activity as examined with N -acetylheparosan oligosaccharides as acceptors.…”

In Vitro Polymerization of Heparan Sulfate Backbone by the EXT Proteins

“…So far no additional GlcA transferases involved in actual HS chain elongation are known. Both EXTL1 and EXTL3 have been shown to be able to transfer GlcNAc (but not GlcA) to K5 oligosaccharide acceptors and are likely to be involved in HS biosynthesis (14). Of the two, only EXTL3 is ubiquitously expressed in human tissues (27,28) and therefore more likely to be part of a universal HS chain-elongating complex.…”

“…The EXTL genes have not been linked to hereditary multiple extoses but belong to the EXT family of proteins based on amino acid sequence similarity to EXT1 and EXT2. All three EXTL proteins express glycosyltransferase activities related to HS biosynthesis; however, their role in HS formation in vivo remains undefined (13,14).…”

A network of clinically and functionally relevant genes is involved in the reversion of the tumorigenic phenotype of MDA-MB-231 breast cancer cells after transfer of human chromosome 8

“…Members of the ADAM protein family have been implicated in cell-cell and cell-matrix interactions (Primakoff and Myles, 2000). EXTL3 is a member of the exostosis (EXT) gene family, which has been tightly associated with glycosyltransferase activities required for the biosynthesis of heparin sulfate (Kim et al, 2001). For two members of the EXT-family, EXT1 and EXT2, a TSG function has been postulated.…”