The link between heparan sulfate and hereditary bone disease: finding a function for the EXT family of putative tumor suppressor proteins (original) (raw)
Human EXT1 and EXT2 (illustrated in Figure 1a) are ubiquitously expressed proteins of 746 and 718 amino acids, respectively, sharing about 70% similarity at the amino acid level (7, 8). EXT1 and EXT2 both have a predicted type II transmembrane glycoprotein structure: a short N-terminal cytoplasmic tail, a transmembrane domain, a stalk region, and a globular lumenal C-terminal tail (17, 18); and each protein localizes predominantly to the endoplasmic reticulum when overexpressed in cell culture (10, 17, 19).
The EXT proteins and HS biosynthesis. (a) Schematic representations of the EXT1 and EXT2 putative tumor suppressor proteins (not drawn to scale). These endoplasmic reticulum–localized (ER-localized) type II transmembrane proteins have an N-terminal cytoplasmic tail, a single transmembrane domain, a stem region, and a long C-terminal lumenal tail. Amino acids (a.a.) mutated in HME patients are labeled and indicated by big stars, amino acids mutated in HS-deficient CHO cells (27) are indicated by small yellow stars, and N-linked glycosylation sites are represented by a pink Y. The proposed D-glucuronic acid glycosyltransferase (GlcA-TII) catalytic domain (27) is shaded in light blue. (b) A schematic representation of the HS biosynthesis pathway (recently reviewed in ref. 43). A tetrasaccharide unit, common to both HS and chondroitin sulfate (CS), is synthesized by sequential additions of xylose (Xyl), two galactose (Gal) residues, and a D-glucuronic acid (GlcA) residue, covalently linked to a serine residue on the HS proteoglycan (HSPG) core protein. HS biosynthesis is specifically initiated by the addition of an _N_-acetylglucosamine residue (GlcNAc), which is carried out by the glycosyltransferase (GlcNAc-TI) encoded by the EXTL2 gene. Next, the HS-polymerase (HS-Pol), a Golgi-localized hetero-oligomeric complex of which EXT1 and EXT2 are key components, elongates the nascent chain by adding alternating GlcA and GlcNAc residues.
Despite extensive genetic characterization, the function 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 biosynthesis (17). Sulfated glycosaminoglycans (GAGs), including 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). Herpes 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 the HS-deficient/HSV-resistant sog9 cell line. After several rounds of screening, a single cDNA was isolated that fully restored HS biosynthesis to the sog9 cell line, thereby rescuing HSV infectivity. Remarkably, DNA sequencing revealed that this cDNA encoded the putative tumor suppressor EXT1 (17) and that the HS-deficient sog9 cell line harbors a specific defect in the EXT1 gene (19). This cell line provides an in vivo functional assay for EXT1 function, because when a functional EXT1 gene is transfected into sog9 cells, the EXT1 defect is complemented, HS synthesis resumes, and the cells regain wild-type levels of susceptibility to HSV (17). This assay is specific for EXT1, because other EXT members are unable to complement the defect in HS biosynthesis (19).
Biochemical studies have confirmed that both EXT1 and its homologue, EXT2, possess the glycosyltransferase activities representative of an HS-polymerase (HS-Pol) in vitro, i.e., the ability to add single D-glucuronic acid (GlcA) and _N_-acetylglucosamine (GlcNAc) molecules to an artificial substrate molecule (18). Another member of the EXT gene family, EXTL2, encodes a functionally related enzyme, α1,4-_N_-acetylhexosaminyltransferase (22). As shown in Figure 1b, EXTL2 is proposed to initiate HS chain formation by transferring the first GlcNAc residue to a specific tetrasaccharide linker sequence on the HSPG core protein, thereby providing the substrate for polymerization by EXT1 and EXT2. By contrast, a study of C. elegans EXT homologues suggests that a single protein, Rib-2, which is most closely related to the human EXTL3 gene product, is able to carry out both the HS chain initiation and the polymerization steps (23), thereby demonstrating that the HS biosynthetic mechanism in C. elegans is distinct from that reported for the mammalian system. A recent report suggests that the human EXTL1 and EXTL3 genes also encode glycosyltransferases involved in HS biosynthesis (24), although another study has suggested that the human EXTL3 gene may encode a molecule with a strikingly different function, a cell surface receptor for the pancreatic β cell regeneration factor Reg (25).
One curious aspect of HME is that mutations in either EXT1 or EXT2 result in the formation of clinically indistinguishable exostoses, even though the two proteins do not appear to be functionally redundant in vivo (19). Insight into this problem came from two observations. First, EXT1 and EXT2 form a hetero-oligomeric complex in vivo that leads to an accumulation of both proteins in the Golgi apparatus (19). Second, Golgi-localized EXT1/EXT2 complexes possess substantially higher glycosyltransferase activity than either EXT1 or EXT2 alone (19). These results suggest a model (Figure 1b) in which a Golgi-localized EXT1/EXT2 heterocomplex represents the biologically relevant form of the HS-Pol enzyme. Solid support for this model has recently been provided by studies in yeast cells, which lack any endogenous HS-Pol activity (26). This model of the HS-Pol heterocomplex provides a convincing explanation of how inherited mutations in either of the two EXT genes might cause loss of HS biosynthesis activity, resulting in clinically identical HME.
More detailed information regarding the glycosyltransferase activity of EXT1 has come from a study involving HS-deficient mutant Chinese hamster ovary (CHO) cells, which were similarly rescued by human EXT1 expression (27). In this system, six reported missense mutations that clustered around a putative nucleotide sugar-binding domain were found to abolish GlcA-transferase (GlcA-T) but not GlcNAc-T activity, suggesting that the GlcA-T catalytic domain lies in the central region of the EXT1 protein.
Two-hybrid analysis has also been used to study the EXT proteins, and results indicate that fragments of the wild-type EXT proteins can interact with a chaperone protein and another glycosyltransferase enzyme. Significantly, these interactions are abrogated by a disease-causing HME mutation (28), suggesting that the EXT proteins may be components of a larger multienzyme GAG synthesis complex.