The link between heparan sulfate and hereditary bone disease: finding a function for the EXT family of putative tumor suppressor proteins Gillian Duncan, … , Craig McCormick, Frank Tufaro J Clin Invest. 2001;108(4):511-516. https://doi.org/10.1172/JCI13737. Perspective Although genetic linkage analysis is a vital tool for identifying disease genes, further study is often hindered by the lack of a known function for the corresponding gene products. In the case of hereditary multiple exostoses (HME), a dominantly inherited genetic disorder characterized by the formation of multiple cartilaginous tumors, extensive genetic analyses of affected families linked HME to mutations in two members of a novel family of putative tumor suppressor genes, EXT1 and EXT2. The biological function of the corresponding proteins, exostosin-1 (EXT1) and exostosin-2 (EXT2), has emerged in part by way of a serendipitous discovery made in the study of herpes simplex virology, which revealed that the pathogenesis of HME is linked to a defect in heparan sulfate (HS) biosynthesis. Biochemical analysis shows that EXT1 and EXT2 are type II transmembrane glycoproteins and form a Golgi-localized hetero-oligomeric complex that catalyzes the polymerization of HS. In this Perspective we will review the identification and characterization of the EXT family, with a particular focus on the biology of the EXT proteins in vivo, and we will explore their possible role(s) in both normal bone development and the formation of exostoses. Hereditary multiple exostoses Hereditary multiple exostoses (HME), an autosomal dominant bone disorder, is the most common type of benign bone tumor, with an estimated occurrence of 1 in 50,000–100,000 in […] Find the latest version: https://jci.me/13737/pdf PERSPECTIVE SERIES Heparan sulfate proteoglycans Renato V. Iozzo, Series Editor The link between heparan sulfate and hereditary bone disease: finding a function for the EXT family of putative tumor suppressor proteins Gillian Duncan, Craig McCormick, and Frank Tufaro Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada Address correspondence to: Frank Tufaro, Department of Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Room 114, Vancouver, British Columbia V6T 1Z3, Canada. Phone: (604) 822-2920; Fax: (604) 822-6041; E-mail: [email protected]. Craig McCormick’s present address is: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, USA. J. Clin. Invest. 108:511–516 (2001). DOI:10.1172/JCI200113737. Although genetic linkage analysis is a vital tool for and restricted movement. Also, recent evidence sug- identifying disease genes, further study is often hin- gests that early removal of exostoses may ameliorate dered by the lack of a known function for the corre- the growth retardation that typically leads to short- sponding gene products. In the case of hereditary mul- ened bones in these individuals (2). While classical tiple exostoses (HME), a dominantly inherited genetic histopathology identifies exostoses as benign disorder characterized by the formation of multiple tumors, the histological organization of an exosto- cartilaginous tumors, extensive genetic analyses of sis shows remarkable similarities with the parent affected families linked HME to mutations in two bone from which it arises. The cartilage cap of an members of a novel family of putative tumor suppres- exostosis often shows the same structural organiza- sor genes, EXT1 and EXT2. The biological function of tion as that of growth plate cartilage, and the tra- the corresponding proteins, exostosin-1 (EXT1) and becular and periosteal bone are continuous with exostosin-2 (EXT2), has emerged in part by way of a that of the underlying bone (1). A neoplastic model serendipitous discovery made in the study of herpes of pathogenesis for exostosis development and pro- simplex virology, which revealed that the pathogenesis gression has recently been put forward as an alter- of HME is linked to a defect in heparan sulfate (HS) native to the traditional skeletal dysplasia theory (3). biosynthesis. Biochemical analysis shows that EXT1 According to this model, which is founded on the and EXT2 are type II transmembrane glycoproteins molecular biology of exostoses, aberrant bone and form a Golgi-localized hetero-oligomeric complex growth would arise through clonal expansion of a that catalyzes the polymerization of HS. In this Per- single mutant chondrocyte, and not due to a defect spective we will review the identification and charac- in all cells of the growth-inducing tissue. Moreover, terization of the EXT family, with a particular focus on some studies have provided evidence that individu- the biology of the EXT proteins in vivo, and we will als with HME might have a significantly higher risk explore their possible role(s) in both normal bone than the general population, 0.5–3%, of developing development and the formation of exostoses. subsequent malignancies such as chondrosarcomas or osteosarcomas. Hereditary multiple exostoses Hereditary multiple exostoses (HME), an autosomal A highly conserved family of putative dominant bone disorder, is the most common type tumor suppressor genes of benign bone tumor, with an estimated occurrence Although the pathogenesis of HME was first described of 1 in 50,000–100,000 in Western populations. It is nearly 200 years ago (1), it is only in the past few years characterized by cartilage-capped tumors, known as that elegant genetic analyses have determined that osteochondromas or exostoses, which develop pri- both hereditary and sporadic cases of exostoses are marily on the long bones of affected individuals linked to two main loci, EXT1 on chromosome 8q24.1 from early childhood until puberty (1). Individuals (4) and EXT2 on chromosome 11p11-p12 (5), with rare with HME are often short in stature with varying linkage to another locus, EXT3, on chromosome 19p degrees of orthopedic deformity. Although exostoses (6). Because exostosis formation is associated with loss are in themselves benign, surgery may be required to of heterozygosity at one or several EXT loci, it has been alleviate secondary complications such as joint pain suggested that the EXT proteins have tumor suppres- The Journal of Clinical Investigation | August 2001 | Volume 108 | Number 4 511 dicted type II transmembrane glycoprotein structure: a short N-terminal cytoplasmic tail, a transmembrane domain, a stalk region, and a globular lumenal C-ter- minal tail (17, 18); and each protein localizes predom- inantly to the endoplasmic reticulum when overex- pressed in cell culture (10, 17, 19). Despite extensive genetic characterization, the func- tion of the EXT proteins remained unknown until 1998, when the study of an HS-deficient cell line, sog9, revealed that EXT1 is involved in HS biosynthe- sis (17). Sulfated glycosaminoglycans (GAGs), includ- ing HS, are negatively charged oligosaccharide chains that decorate cell surface and ECM proteoglycans (PGs), playing important roles in ligand-binding, cell adhesion, and cell signaling (reviewed in ref. 20). Her- pes simplex viruses (HSVs), like many other enveloped viruses, use HS as a primary receptor for attachment to the host cell (See Shukla and Spear, this Perspective series, ref. 21). Our laboratory screened for cDNAs capable of restoring susceptibility to HSV infection in Figure 1 the HS-deficient/HSV-resistant sog9 cell line. After The EXT proteins and HS biosynthesis. (a) Schematic representations of several rounds of screening, a single cDNA was isolat- the EXT1 and EXT2 putative tumor suppressor proteins (not drawn to ed that fully restored HS biosynthesis to the sog9 cell scale). These endoplasmic reticulum–localized (ER-localized) type II trans- line, thereby rescuing HSV infectivity. Remarkably, membrane proteins have an N-terminal cytoplasmic tail, a single trans- DNA sequencing revealed that this cDNA encoded membrane domain, a stem region, and a long C-terminal lumenal tail. Amino acids (a.a.) mutated in HME patients are labeled and indicated by the putative tumor suppressor EXT1 (17) and that the big stars, amino acids mutated in HS-deficient CHO cells (27) are indicat- HS-deficient sog9 cell line harbors a specific defect in ed by small yellow stars, and N-linked glycosylation sites are represented by the EXT1 gene (19). This cell line provides an in vivo a pink Y. The proposed D-glucuronic acid glycosyltransferase (GlcA-TII) cat- functional assay for EXT1 function, because when a alytic domain (27) is shaded in light blue. (b) A schematic representation functional EXT1 gene is transfected into sog9 cells, of the HS biosynthesis pathway (recently reviewed in ref. 43). A tetrasac- the EXT1 defect is complemented, HS synthesis charide unit, common to both HS and chondroitin sulfate (CS), is synthe- resumes, and the cells regain wild-type levels of sus- sized by sequential additions of xylose (Xyl), two galactose (Gal) residues, ceptibility to HSV (17). This assay is specific for EXT1, and a D-glucuronic acid (GlcA) residue, covalently linked to a serine residue because other EXT members are unable to comple- on the HS proteoglycan (HSPG) core protein. HS biosynthesis is specifi- ment the defect in HS biosynthesis (19). cally initiated by the addition of an N-acetylglucosamine residue (GlcNAc), which is carried out by the glycosyltransferase (GlcNAc-TI) encoded by the Biochemical studies have confirmed