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UBC Theses and Dissertations

Multiple exostoses gene, EXT1 and heparan sulfate biosynthesis Cheung, Peter Kwai-yin


Hereditary multiple exostoses (HME), a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 and EXT2. The corresponding gene products, exostosin-1 (EXT1) and exostosin-2 (EXT2) are type II transmembrane glycoproteins which form a Golgi-Iocalized hetero-oligomeric complex that catalyzes the polymerization of heparan sulfate (HS). Although the majority of the etiological mutations in EXT are splice-site, frameshift or nonsense mutations that result in premature termination, a significant number of missense mutations have also been identified. To test the etiological missense mutant of the EXT1 proteins for their ability to synthesize HS in vivo, a functional assay that detects HS expression on the cell surface of an EXT 1-deficient cell line was used. Of the twelve reported missense mutations, eight were defective in HS biosynthesis, but surprisingly, four were phenotypically indistinguishable from wild type EXT1. Three of these four phenotypically wild type EXT1 like mutations affected amino acids that were non-conserved among vertebrates and invertebrates, whereas all of the HS biosynthesis null mutations affected only conserved amino acids. Further, substitution or deletion of each of these four residues did not abrogate HS biosynthesis. Taken together, these results indicated that not all reported etiological missense mutations abrogated HS-Pol activity. These mutations may instead interfere with as yet undefined functions of EXT involved in HME pathogenesis. The fact that HME is an autosomal dominant genetic disorder suggests that one mutant copy of the gene should be sufficient to cause disease. However, no dominant negative phenotype was observed, even when mutant and wild-type forms of EXT1 proteins were cotransfected in a ratio of 10:1. Furthermore, the two adjacent amino acid residues G339 and R340, which account for five out of twelve known etiological missense mutations of the EXT1 gene, were localized in a consensus cleavage site for furin, a proprotein convertase. Results from both in vitro and in vivo experiments suggested that the putative cleavage sites were not used and EXT1 protein was expressed in its full-length form. During the course of this study, nine HS deficient cell lines were isolated based on an HSV-1 resistant phenotype. Surprisingly, EXT1 alone corrected the HS deficiency of all nine mutant cell lines, although four other EXT genes (EXT2, EXTL1, EXTL2 and EXTL3) have now been identified and likely harbour glycosyltransferase activities that contribute to the synthesis of HS. These observations suggest that some glycosyltransferases which are involved in HS biosynthesis may be essential for cell survival and/or may exist in multiple copies. Because none of the nine HS deficient cell lines have detectable defects in EXT2, and only EXT1 corrects the HS deficiency, EXT1 could be the sole enzyme that polymerizes HS in mammalian cells.

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