Frank Tufaro

The Putative Tumor Suppressors EXT1 and EXT2 Are Glycosyltransferases Required for the Biosynthesis of Heparan Sulfate

Hereditary multiple exostoses, characterized by multiple cartilaginous tumors, is ascribed to mutations at three distinct loci, denoted EXT1–3. Here, we report the purification of a protein from bovine serum that harbored thed-glucuronyl (GlcA) andN-acetyl-d-glucosaminyl (GlcNAc) transferase activities required for biosynthesis of the glycosaminoglycan, heparan sulfate (HS). This protein was identified as EXT2. Expression of EXT2 yielded a protein with both glycosyltransferase activities. Moreover, EXT1, previously found to rescue defective HS biosynthesis (McCormick, C., Leduc, Y., Martindale, D., Mattison, K., Esford, L. E., Dyer, A. P., and Tufaro, F. (1998) Nat. Genet. 19, 158–161), was shown to elevate the low GlcA and GlcNAc transferase levels of mutant cells. Thus at least two members of the EXT family of tumor suppressors encode glycosyltransferases involved in the chain elongation step of HS biosynthesis.

The putative tumour suppressor EXT1 alters the expression of cell-surface heparan sulfate

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by the formation of cartilage-capped tumours (exostoses) that develop from the growth plate of endochondral bone. This condition can lead to skeletal abnormalities, short stature and malignant transformation of exostoses to chondrosarcomas or osteosarcomas. Linkage analyses have identified three different genes for HME, EXT1 on 8q24.1, EXT2 on 11p11–13 and EXT3 on 19p (refs 6, 7, 8, 9). Most HME cases have been attributed to missense or frameshift mutations in these tumour-supressor genes, whose functions have remained obscure. Here, we show that EXT1 is an ER-resident type II transmembrane glycoprotein whose expression in cells results in the alteration of the synthesis and display of cell surface heparan sulfate glycosaminoglycans (GAGs). Two EXT1 variants containing aetiologic missense mutations failed to alter cell-surface glycosaminoglycans, despite retaining their ER-localization.

The link between heparan sulfate and hereditary bone disease: finding a function for the EXT family of putative tumor suppressor proteins

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 Western populations. It is

Chondroitin 4-O-sulfotransferase -1 regulates E disaccharide expression of chondroitin sulfate required for herpes simplex virus infectivity

We have demonstrated a defect in expression of chondroitin 4-O-sulfotransferase-1 (C4ST-1) in murine sog9 cells, which are poorly sensitive to infection by herpes simplex virus type 1 (HSV-1). Sog9 cells were previously isolated as CS-deficient cells from gro2C cells, which were partially resistant to HSV-1 infection and defective in the expression of heparan sulfate (HS) because of a splice site mutation in the EXT1 gene encoding the HS-synthesizing enzyme. Here we detected a small amount of CS chains in sog9 cells with a drastic decrease in 4-O-sulfation compared with the parental gro2C cells. RT-PCR revealed that sog9 cells had a defect in the expression of C4ST-1 in addition to EXT1. Gel filtration analysis showed that the decrease in the amount of CS in sog9 cells was the result of a reduction in the length of CS chains. Transfer of C4ST-1 cDNA into sog9 cells (sog9-C4ST-1) restored 4-O-sulfation and amount of CS, verifying that sog9 cells had a specific defect in C4ST-1. Furthermore, the expression of C4ST-1 rendered sog9 cells significantly more susceptible to HSV-1 infection, suggesting that CS modified by C4ST-1 is sufficient for the binding and infectivity of HSV-1. Analysis of CS chains of gro2C and sog9-C4ST-1 cells revealed a considerable proportion of the E disaccharide unit, consistent with our recent finding that this unit is an essential component of the HSV receptor. These results suggest that C4ST-1 regulates the expression of the E disaccharide unit and the length of CS chains, the features that facilitate infection of cells by HSV-1.

Herpes simplex virus type 1 glycoprotein C is necessary for efficient infection of chondroitin sulfate-expressing gro2C cells.

The role of glycoprotein C (gC) for binding of herpes simplex virus type 1 (HSV-1) to cell surface chondroitin sulfate (CS) and the consequences of this interaction for virus attachment and infectivity were studied. To this end, a panel of HSV-1 gC mutants, including a gC-negative (gC(-)) variant, and mouse fibroblasts expressing either cell surface CS or heparan sulfate (HS) were used. Comparing gC-positive (gC(+)) and gC(-) viruses in terms of their attachment to and infection of CS-expressing cells indicated that gC was essential for both functions. Furthermore, purified gC bound efficiently to isolated CS chains. However, hypertonic NaCl disrupted this interaction more easily as compared to the binding of gC to HS. Also, native and selectively desulfated heparins were approximately 10 times more efficient at inhibiting gC binding to CS-expressing cells than binding to HS-expressing cells. Experiments with the HSV-1 gC mutants revealed that specific, positively charged and hydrophobic amino acids within the N-terminal part of the protein were responsible for efficient binding as well as infectivity in both CS- and HS-expressing cells. When the infectivity of the gC mutants in the two cell types was compared, it appeared that more residues contributed to the infection of CS-expressing cells than to infection of HS-expressing cells. Taken together, analysis of gC function in cell systems with limited expression of glycosaminoglycans revealed that gC could interact with either CS or HS and that these interactions exhibited subtle but definite differences as regards to the involved structural features of gC, ionic strength dependency as well as sensitivity to specifically desulfated heparin compounds.

Neutralization of Cytomegalovirus Virions: The Role of Complement

Complement provides a key immunologic defense against invading pathogens; thus, a clear understanding of the interactions between cytomegalovirus (CMV) and complement may permit the development of strategies to enhance CMV neutralization. In the presence of specific anti-CMV antibodies, complement enhanced the neutralizing ability of serum by 2- to 3-fold. However, in the absence of specific anti-CMV antibodies, complement was ineffective in neutralizing CMV virions by plaque assay. Although complement alone did not mediate any neutralizing effect, CMV consumed complement activity from seronegative serum, resulting in the deposition of C3 on the virion. However, only in the presence of specific anti-CMV antibody did complement activation continue to the deposition of C9 on the virions. These results strongly suggest complement regulation by CMV virions that is modulated by anti-CMV antibody; this regulation may be attributed to three host complement regulators on the virions: CD55, CD46, and CD59.

A bcl-2 expressing viral vector protects cortical neurons from excitotoxicity even when administered several hours after the toxic insult

The product of the bcl-2 oncogene has been shown to play an important role in apoptosis and programmed cell death. In this study, a herpes simplex virus type-1 vector was constructed to carry the human bcl-2 gene. The possible role of bcl-2 in protecting neurons from excitoxicity was investigated by using the viral vector to deliver the gene into neuronal cultures before or after the cells were exposed to glutamate under conditions in which 50-80% of neurons died. Infection with the bcl-2 expressing vector 24 h prior to glutamate treatment effectively prevented the cell death that normally follows this treatment. Moreover, infection with the vector as late as 8 h after the glutamate insult still resulted in substantial neuroprotective effects. These results have potential implications for new therapies in stroke or ischemic neuropathies.

New perspectives on the molecular basis of hereditary bone tumours

Bone development is a highly regulated process sensitive to a wide variety of hormones, inflammatory mediators and growth factors. One of the most common hereditary skeletal dysplasias, hereditary multiple exostoses (HME), is an autosomal dominant disorder characterized by skeletal malformations that manifest as bony, benign tumours near the end of long bones. HME is usually caused by defects in either one of two genes, EXT1 and EXT2, which encode enzymes that catalyse the biosynthesis of heparan sulphate, an important component of the extracellular matrix. Thus, HME-linked bone tumours, like many other skeletal dysplasias, probably result from disruptions in cell surface architecture. However, despite the recent success in unravelling functions for several members of the EXT gene family, significant challenges remain before this knowledge can be used to develop new approaches for the diagnosis and treatment of disease.

The varicella zoster virus glycoprotein B (gB) plays a role in virus binding to cell surface heparan sulfate proteoglycans

Varicella-zoster virus (VZV) interacts with cell surface heparan sulfate proteoglycans during virus attachment. In the present study, we investigated the potential involvement of two VZV glycoproteins, gB and gE, in the virus adsorption process. We showed that gB, but not gE, binds specifically to cellular heparan sulfate proteoglycans (HSPGs). Indeed, soluble recombinant gB protein (recgB) was found to bind to immobilized heparin and to MRC5 and L cells, a binding which was inhibited by heparin. Furthermore, recgB binding to two heparan sulfate-minus mutant L cell lines, gro2C and sog9 cells, was markedly reduced as compared to the parental L strain. Under the same experimental conditions, soluble recombinant VZV gE protein did not interact with heparin or with cell surfaces. We also demonstrated that the gB-HSPGs interactions were relevant to the VZV attachment to cells. Indeed, although polyclonal antibodies directed to gB did not impair the VZV binding, recgB could delay the virus adsorption. Our results thus strongly suggest that the interactions between gB and heparan sulfate proteoglycans take part in the initial VZV attachment to cell surfaces.

A Perspective of Gene Therapy in the Glaucomas

Gene therapy in the anterior and posterior segment tissues may have the potential to favorably influence aqueous hydrodynamics and retinal ganglion cell biology, thereby preventing, delaying, or minimizing glaucomatous damage to the optic nerve. We demonstrated the feasibility of using a herpes viral vector (ribonucleotide reductase defective HSV-1, hrR3) to deliver the lacZ reporter gene to living cat and rat eyes. Cats received injections into the anterior chamber and rats into the vitreous cavity. In cats, lacZ expression was detectable at 1 to 2 days in the anterior outer portion of the ciliary muscle and the lining of the intertrabecular spaces of the corneoscleral and uveal meshwork. Rat eyes showed lacZ expression in the retinal pigment epithelium and photoreceptor outer segments 2 days after injection.