The putative tumor suppressors EXT1 and EXT2 form a stable complex that accumulates in the Golgi apparatus and catalyzes the synthesis of heparan sulfate

Abstract

Hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The proteins encoded by these genes, EXT1 and EXT2, are endoplasmic reticulum-localized type II transmembrane glycoproteins that possess or are tightly associated with glycosyltransferase activities involved in the polymerization of heparan sulfate. Here, by testing a cell line with a specific defect in EXT1 in in vivo and in vitro assays, we show that EXT2 does not harbor significant glycosyltransferase activity in the absence of EXT1. Instead, it appears that EXT1 and EXT2 form a hetero-oligomeric complex in vivo that leads to the accumulation of both proteins in the Golgi apparatus. Remarkably, the Golgi-localized EXT1͞EXT2 complex possesses substantially higher glycosyltransferase activity than EXT1 or EXT2 alone, which suggests that the complex represents the biologically relevant form of the enzyme(s). These findings provide a rationale to explain how inherited mutations in either of the two EXT genes can cause loss of activity, resulting in hereditary multiple exostoses. Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by the formation of cartilagecapped tumors (exostoses) that develop from the growth plate of endochondral bone (1). This condition can lead to skeletal abnormalities, short stature, and in some instances, malignant transformation from exostoses to chondrosarcomas (2, 3) or osteosarcomas (4, 5). Although genetic linkage analysis has identified three different loci for HME, EXT1 on 8q24.1, EXT2 on 11p11-13, and EXT3 on 19p (6-8), most HME cases have been attributed to missense or frameshift mutations in either EXT1 or EXT2 (9-15). EXT1 and EXT2 encode 746-and 718-aa proteins, respectively, that are expressed ubiquitously in human tissues (9, 16).Previous studies using epitope-tagged constructs have demonstrated that EXT1 is a predominantly endoplasmic reticulum (ER)-localized glycoprotein whose expression enhances the synthesis of cell surface heparan sulfate (HS) (17). HS chains are composed of alternating residues of D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc) joined by 134 linkages, and a recent study has shown that both EXT1 and EXT2 harbor GlcA transferase (GlcA-T) and GlcNAc transferase (GlcNAc-T) activities that catalyze the polymerization of HS (18). EXT1 and EXT2 are structurally similar to previously identified glycosyltransferases in that they are type II transmembrane proteins comprising an N-terminal cytoplasmic tail, a transmembrane domain, a stalk, and a large globular domain that is likely to harbor enzymatic activity (19). Moreover, a truncated active form of EXT2 is secreted from cells and can be isolated from serum (18), which is a fate common to other ER and Golgilocalized glycosyltransferases, including EXTL2, which is an EXT homolog shown to encode an ␣1,4-N-acetylhexosaminyltransferase (20). However, several important questions are...

Functional conservation of the human EXT1 tumor suppressor gene and its Drosophila homolog tout velu

“…Human EXT1-Green Fluorescent Protein (hEXT1-GFP) was polymerase chain reaction amplified from pEXT1 GFP (McCormick et al 2000; a gift from Tufaro lab, University of British Columbia), harboring GFP downstream of EXT1 gene, using the primers 5′-GGA CTC AGA TCC CGC AGG ACA CAT-3′ and 5′-CCT CTA CAAATG TGG TAT GGC TGA TTATGA-3′ and cloned into pGEM-T-Easy (Invitrogen) and pUASp2 (a gift from Pernille Rorth) vectors at EcoRI site. The sotv complementary DNA (cDNA) cloned in pAC5.1/V5-His C construct (Han et al 2004b, a gift from Xinhua Lin's lab).…”

“…In both Drosophila and mammals, EXT1 or EXT2 protein localizes mainly in the ER (McCormick et al , 2000The et al 1999;Han et al 2004b;Kobayashi et al 2000). However, when both the proteins were expressed in the same cell, the EXT proteins were found to be mostly relocated from the ER to the golgi network (McCormick et al 2000) where polymerization and sulfation of HSPG occurs (Muckenthaler et al 1998).…”

“…However, when both the proteins were expressed in the same cell, the EXT proteins were found to be mostly relocated from the ER to the golgi network (McCormick et al 2000) where polymerization and sulfation of HSPG occurs (Muckenthaler et al 1998). To determine the subcellular localization of hEXT1-GFP in Drosophila, the wing imaginal discs expressing hEXT1-GFP under the control of en-GAL4 were stained for colocalization with plasma membrane marker dye FM4-64 (Fig.…”

“…The EXT genes encode for type II transmembrane glycoproteins that localize predominantly to the endoplasmic reticulum (ER) and golgi. They form a heterooligomeric complex in the golgi apparatus (Kobayashi et al 2000;McCormick et al 2000) and are involved in the synthesis of heparan sulfate glycosaminoglycans (HSGAGs; Lind et al 1998;McCormick et al 1998McCormick et al , 2000Toyoda et al 2000b;Wei et al 2000). Heparan sulfate proteoglycans (HSPGs) consist of a glycosylated protein core, which can either be a transmembrane, glycosylphosphatidyl-inositol (GPI)-linked, or secreted protein, and they are abundant on the cell surface and extracellular matrix.…”

Epiphyseal growth plate and secondary peripheral chondrosarcoma: the neighbours matter

“…EXT1 and EXT2 encode type II transmembrane glycosyltransferases [45,46], whose functions are not fully known. EXT1 and EXT2 form a hetero-oligomeric complex in the Golgi apparatus of most human cells that participate in chain elongation in heparan sulphate biosynthesis [47,48]. Albeit the genetic correlation between mutations in EXT1/EXT2 and osteochondromas, the mechanism by which alterations in heparan sulphate biosynthesis leads to osteochondroma is not entirely understood.…”