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The Putative Tumor Suppressors EXT1 and EXT2 Form a Stable Complex That Accumulates in the Golgi Apparatus and Catalyzes the Synthesis of Heparan Sulfate

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The Putative Tumor Suppressors EXT1 and EXT2 Form a Stable Complex That Accumulates in the Golgi Apparatus and Catalyzes the Synthesis of Heparan Sulfate The putative tumor suppressors EXT1 and EXT2 form a stable complex that accumulates in the Golgi apparatus and catalyzes the synthesis of heparan sulfate Craig McCormick, Gillian Duncan, K. Tina Goutsos, and Frank Tufaro* Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada Edited by Kai Simons, European Molecular Biology Laboratory, Heidelberg, Germany, and approved November 19, 1999 (received for review August 23, 1999) Hereditary multiple exostoses, a dominantly inherited genetic raised by these observations. EXT1, when overexpressed in a disorder characterized by multiple cartilaginous tumors, is caused cell, appears to be localized predominantly to the ER (17, 21), by mutations in members of the EXT gene family, EXT1 or EXT2. The whereas the biosynthesis of HS chains is thought to occur in the proteins encoded by these genes, EXT1 and EXT2, are endoplasmic Golgi cisternae (22–25). Moreover, if the EXT1 and EXT2 genes reticulum-localized type II transmembrane glycoproteins that pos- encode functionally redundant HS polymerases (HS-Pol), it is sess or are tightly associated with glycosyltransferase activities not clear why mutations in either gene cause HME. involved in the polymerization of heparan sulfate. Here, by testing To address these questions, we overexpressed functional a cell line with a specific defect in EXT1 in in vivo and in vitro assays, epitope-tagged and native forms of EXT1 and EXT2 in cells and we show that EXT2 does not harbor significant glycosyltransferase examined their subcellular localization and enzymatic activity. activity in the absence of EXT1. Instead, it appears that EXT1 and By using a cell line, sog9, with a specific defect in EXT1, we show EXT2 form a hetero-oligomeric complex in vivo that leads to the that EXT2 does not harbor significant glycosyltransferase activ- accumulation of both proteins in the Golgi apparatus. Remarkably, ity in the absence of EXT1. Instead, it appears that EXT1 and the Golgi-localized EXT1͞EXT2 complex possesses substantially EXT2 form a hetero-oligomeric complex in vivo that leads to an higher glycosyltransferase activity than EXT1 or EXT2 alone, which accumulation of both proteins in the Golgi apparatus. Remark- suggests that the complex represents the biologically relevant ably, the Golgi-localized EXT1͞EXT2 complex possesses sub- form of the enzyme(s). These findings provide a rationale to stantially higher glycosyltransferase activity than EXT1 or EXT2 explain how inherited mutations in either of the two EXT genes can alone, which suggests that this complex represents the biologi- cause loss of activity, resulting in hereditary multiple exostoses. cally relevant form of the enzyme(s). These findings provide a rationale to explain how inherited mutations in either of the two ereditary multiple exostoses (HME) is an autosomal dom- EXT genes can cause loss of activity, resulting in hereditary Hinant disorder characterized by the formation of cartilage- multiple exostoses. capped tumors (exostoses) that develop from the growth plate of Materials and Methods endochondral bone (1). This condition can lead to skeletal EXT Constructs. pEXT1 was isolated from a HeLa cell cDNA abnormalities, short stature, and in some instances, malignant library in pcDNA3.1 (A550–26, Invitrogen) as described previ- transformation from exostoses to chondrosarcomas (2, 3) or ously (17). pEXT1 myc-His, pG339DEXT1 myc-His, and osteosarcomas (4, 5). Although genetic linkage analysis has pR340CEXT1 myc-His were constructed as described previously identified three different loci for HME, EXT1 on 8q24.1, EXT2 (17). All reagents were obtained from Life Technologies unless on 11p11–13, and EXT3 on 19p (6–8), most HME cases have otherwise stated. pEXT1 GFP was constructed by excision of been attributed to missense or frameshift mutations in either EXT1 from pEXT1 myc-His with BamHI and SstII, followed by EXT1 or EXT2 (9–15). EXT1 and EXT2 encode 746- and 718-aa ligation into the BglII and SstII sites in the pEGFP-N1 expression proteins, respectively, that are expressed ubiquitously in human vector (CLONTECH). The bovine EXT2 constructs were con- tissues (9, 16). structed as previously described (18). pbEXT2 myc was con- Previous studies using epitope-tagged constructs have dem- structed by PCR of the EXT2 coding region by using primers onstrated that EXT1 is a predominantly endoplasmic reticulum 5Ј-CGG GAT CCC GGT TTC ATT ATG TGT GCG TCA GTC (ER)-localized glycoprotein whose expression enhances the AAG TCC AAC A-3Ј and 5Ј-GCT CTA GAG CTC ACA GAT synthesis of cell surface heparan sulfate (HS) (17). HS chains are CCT CTT CTG AGA TGA GTT TTT GTT CTA AGC TGC composed of alternating residues of D-glucuronic acid (GlcA) Ј 3 CAA TGT TGG-3 . After digestion with BamHI and XbaI, the and N-acetyl-D-glucosamine (GlcNAc) joined by 1 4 linkages, bEXT2 myc PCR product was then ligated into pcDNA3.1͞myc- and a recent study has shown that both EXT1 and EXT2 harbor His B. A murine EXT2 (mEXT2) cDNA was a gift from M. GlcA transferase (GlcA-T) and GlcNAc transferase (GlcNAc-T) Lovett (Washington University School of Medicine, St. Louis). activities that catalyze the polymerization of HS (18). EXT1 and EXT2 are structurally similar to previously identified glycosyl- transferases in that they are type II transmembrane proteins This paper was submitted directly (Track II) to the PNAS office. comprising an N-terminal cytoplasmic tail, a transmembrane Abbreviations: HME, hereditary multiple exostoses; ER, endoplasmic reticulum; HS, hepa- domain, a stalk, and a large globular domain that is likely to ran sulfate; GlcA, D-glucuronic acid; GlcNAc, N-acetyl-D-glucosamine; GlcA-T, glucuronyl- harbor enzymatic activity (19). Moreover, a truncated active transferase; GlcNAc-T, N-acetyl-D-glucosaminyltransferase; HS-Pol, heparan sulfate poly- merase; HSV-1, herpes simplex virus type I; GFP, green fluorescent protein; endoH, en- form of EXT2 is secreted from cells and can be isolated from doglycosidase H. serum (18), which is a fate common to other ER and Golgi- *To whom reprint requests should be addressed. E-mail: [email protected]. localized glycosyltransferases, including EXTL2, which is an ␣ The publication costs of this article were defrayed in part by page charge payment. This EXT homolog shown to encode an 1,4-N-acetylhexosaminyl- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. transferase (20). However, several important questions are §1734 solely to indicate this fact. 668–673 ͉ PNAS ͉ January 18, 2000 ͉ vol. 97 ͉ no. 2 Downloaded by guest on September 26, 2021 pmEXT2 GFP was constructed by PCR using primers 5Ј-CGG Immunoprecipitations. BHK cells (1 ϫ 106) were transfected with GAT CCC GGT TTC ATT ATG TGT GCG TCA GTC AAG green fluorescent protein (GFP) or Myc-His-tagged EXT con- TCC AAC A-3Ј and 5Ј-TCC CCG CGG GGA TAA GCT GCC structs. After 20 h, cells were radiolabeled with 100 ␮Ci͞ml (1 AAT GTT GGG GAA-3Ј. The mEXT2 PCR product was ␮Ci ϭ 37 kBq) [35S]methionine (ICN) in methionine- and ligated into T-tailed pBluescript (Stratagene), followed by di- cysteine-free DMEM (ICN) for 1.5 h at 37°C. Cells were washed gestion with HindIII and EcoRI and subsequent ligation into with PBS and lysed in Triton lysis buffer [2% Triton X-100͞20 ⅐ ͞ pEGFP-N1. pmEXT2 myc-His was constructed by digestion of mM Tris HCl pH 7.4 150 mM NaCl containing CØmplete pmEXT2 GFP with HindIII and SstII and ligation into the protease inhibitors (Roche)] at 4°C for 15 min. The lysates were ϫ pcDNA3.1͞myc-His B vector. To isolate human EXTL2, centrifuged at 12,000 g for 15 min, and precleared for 30 min ␮ EXTL3, and murine N-deacetylase͞N-sulfotransferase-2 with 25 l of protein G-Sepharose (Pharmacia) at 4°C. The lysates were then incubated with 0.5 ␮g of mouse anti-Myc (NDST2), total cellular RNA was isolated from confluent HeLa ␮ cell and L cell cultures by using Trizol reagent, reverse tran- monoclonal antibody (Invitrogen) or 0.5 g of rabbit anti-GFP scribed and amplified by PCR using primers 5Ј-CCG CTC GAG monoclonal antibody (CLONTECH) for 2 h, followed by incu- Ј Ј bation with 25 ␮l of protein G-Sepharose for 1 h. The lysates CGG AAT TAA ACT TCA ACA CAA TG-3 and 5 -GGG ϫ Ј were centrifuged at 12,000 g for 10 s, and washed two times GTA CCC CTA TTT TTC TTT TGT AGT TGG CAT-3 for ⅐ ͞ ͞ ͞ Ј with 10 mM Tris HCl pH 7.4 150 mM NaCl 2 mM EDTA 0.2% EXTL2,5-CCG CTC GAG CGG CAG GCT GCA GAG GAC ⅐ ͞ Ј Ј Triton X-100, two times with 10 mM Tris HCl, pH 7.4 500 mM TCA T-3 and 5 -GGG GTA CCC CGA TGA ACT TGA AGC ͞ ͞ Ј Ј NaCl 2 mM EDTA 0.2% Triton X-100, and two times with 10 ACT TGG TCT-3 for EXTL3 and 5 -GAA GAT CTT CCC mM Tris⅐HCl, pH 7.4. The pellet was suspended in 30 ␮lof ACC ATG CTC CAG CTG TGG AAG GT-3Ј and 5Ј-CGG ͞ ͞ Ј SDS PAGE sample buffer and boiled for 5 min before SDS AAT TCC GCC CAC ACT GGA ATG TTG CAA T-3 for PAGE. Proteins were transferred to Immobilon-P membranes NDST2. The digested PCR products were ligated into the (Millipore) and exposed to BioMAX MR film (Kodak). appropriate sites in pEGFP-N1. G339DEXT1-GFP, ⌬ R340CEXT1-GFP, and NTMEXT1-GFP were constructed by Assay of Cellular Glycosyltransferase Activities. BHK or mutant digestion and subsequent ligation into pEGFP-N1. sog9 cells were transfected with EXT constructs. At 30 h after transfection, cells were washed in PBS and lysed in Triton͞ Herpes Simplex Virus Type 1 (HSV-1) Infection Assay.
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