Characterization of heparan sulphate 3-O-sulphotransferase isoform 6 and its role in assisting the entry of herpes simplex virus type 1

Abstract

Heparan sulphate (HS) 3-O-sulphotransferase transfers sulphate to the 3-OH position of the glucosamine residue of HS to form 3-O-sulphated HS. The HS modified by 3-O-sulphotransferase isoform 3 binds to HSV-1 (herpes simplex virus type 1) gD (envelope glycoprotein D), and the resultant 3-O-sulphated HS serves as an entry receptor for HSV-1. In the present paper, we report the isolation and characterization of a novel HS 3-O-sulphotransferase isoform, designated HS 3-O-sulphotransferase isoform 6 (3-OST-6). Mouse 3-OST-6 gene was identified in the EST (expressed sequence tag) database and cloned into pcDNA3.1/Myc-His vector. A CHO (Chinese-hamster ovary) cell line that stably expresses 3-OST-6 (3OST6/CHO cells) was prepared. The disaccharide analysis of the HS isolated from 3OST6/CHO cells revealed that 3-OST-6 exhibits HS 3-O-sulphotransferase activity. Furthermore, 3OST6/CHO cells were susceptible to infection by HSV-1, but not by other alphaherpesviruses examined, suggesting that 3-OST-6 produces a specific entry receptor for HSV-1. Our results indicate that a new member of 3-OST family generates an entry receptor for HSV-1. The findings add to the growing body of evidence that HSV-1 entry is mediated by 3-O-sulphated HS generated by multiple members of 3-O-sulphotransferases.

Figure 1. Substrate specificity of 3-OSTs and the structure of HS disaccharide repeating unit

3-OST-1 transfers sulphate to the 3-OH position of a glucosamine N-sulphate (GlcNS±6S) unit that is linked to a GlcA unit to form the 3-O-sulphated HS containing GlcA-GlcNS3S±6S, which is an anticoagulant HS. 3-OST-3 transfers sulphate to the 3-OH position of an N-unsubstituted glucosamine unit (GlcNH₂±6S) that is linked to an IdoA2S unit to form the 3-O-sulphated HS containing IdoA2S-GlcNH₂3S±6S, which is an entry receptor for HSV-1. Both 3-OST-3A and 3-OST-3B have nearly identical amino acid sequences in the sulphotransferase domain, and sulphate identical disaccharides. For clarity, 3-OST-3 represents both 3-OST-3A and 3-OST-3B, unless specified. 3-OST-5 transfers sulphate to the 3-OH position of a modified glucosamine unit (GlcNH₂±6S or GlcNS±6S) that is linked to various GlcA or IdoA/IdoA2S units. The HS modified by 3-OST-5 displays anticoagulant activity and serves as entry receptor for HSV-1. The numbers indicate the positions of saccharide unit. The 3-O-sulphation by 3-OSTs is shaded and indicated in bold. R represents a proton (-H) or a sulphate group (-SO₃).

Figure 2. Nucleotide and deduced amino acid sequences of mouse 3-OST-6

The single predicted membrane-spanning domain and a potential N-linked glycosylation site are indicated by the double underlining and by the underlining with a • below the glycosylated asparagine residue respectively.

Figure 3. Multiple amino acid sequence alignment of mouse 3-OST-6 with mouse 3-OST-1, 3-OST-3A and 3-OST-5

The alignment was performed by using the BioEdit program. Introduced gaps are shown as dashes, and aligned amino acids are boxed and shaded black for identical residues and dark grey for similar residues. 5′-PBS represents the putative domain that binds to the 5′-phosphate of PAPS, and 3′-PBS represents the putative domain that binds to the 3′-phosphate of PAPS.

Figure 4. Western blotting analysis of 3OST6/CHO cell extract

3OST6/CHO and CHO-K1 cell extracts were resolved on SDS/13% PAGE. Myc-tagged 3-OST-6 was detected by incubating the blot with mouse anti-Myc antibody and then horseradishperoxidase-conjugated anti-mouse IgG secondary antibody. The apparent molecular masses of the two bands were approx. 38 kDa and 41 kDa. The predicted molecular mass of 3-OST-6–Myc/His is 37.4 kDa. Molecular masses of standards are given to the right of the gel.

Figure 5. RPIP-HPLC chromatograms of the disaccharide analysis of HS isolated from 3OST6/CHO cells

[³⁵S]HS was extracted from CHO-K1 (A) and 3OST6/CHO (B) cells after incubating them with Na₂³⁵SO₄. [³⁵S]HS was then depolymerized by nitrous acid at pH 1.5, followed by sodium borohydride reduction. The resultant ³⁵S-labelled disaccharides were resolved on RPIP-HPLC. The elution positions of disaccharides are indicated by arrows, where arrow 1 represents IdoA2S-AnMan3S, arrow 2 represents IdoA2S-AnMan6S, and arrow 3 represents IdoA2S-AnMan3S6S. (C) The reaction used for degrading HS with nitrous acid. Nitrous acid (at pH 1.5) reacts with an N-sulphated glucosamine (GlcNSO₃) unit to form 2,5-anhydromannitol (AnMan). The identities of the resultant disaccharides were determined by co-eluting with disaccharide standards on RPIP-HPLC.

Figure 6. Entry of HSV-1 into 3OST6/CHO cells and CHO-K1 cells

(A) As indicated, 3OST6/CHO, 3OST5/CHO and CHO-K1 cells were infected with several PFUs (plaque-forming units)/well of HSV-1(KOS-gL86). The cells were lysed for the quantification of β-galactosidase activity as a measure of viral entry. Attenuance at 410 nm (OD 410 nm) of ONPG (o-nitrophenyl β-D-galactopyranoside) reaction products were plotted against PFUs used. 3OST6/CHO (B) and CHO-K1 (C) cells were exposed to KOS-gL86 at 100 (PFUs)/cell. At 6 h later, the cells were washed, fixed and incubated with X-gal (5-bromo-4-chloroindol-3-yl β-D-galactopyranoside) to identify infected cells (dark cells).

Figure 7. Cell–cell fusion mediated by 3-O-sulphated HS

(A) Cell fusion is dependent on expression of 3-OST-6. CHO-K1 cells were used as effector and target cell. Effector cells were transfected with plasmid expressing HSV-1 glycoproteins and luciferase reporter plasmids. Target cells were transfected with T7 RNA polymerase and the plasmid expressing 3-OST-5 or 3-OST-6. Luciferase activity was measured 24 h after mixing and co-cultivating the effector and target cells. The luciferase activity is from one experiment performed in triplicate. (B) and (C) are the microscopic images for the observation of HSV-1 glycoprotein-induced cell fusion. CHO-K1 effector cells were transfected with HSV-1 glycoproteins (gB, gD, gH and gL). The target CHO-K1cells were transfected either with an empty vector (B) or with the plasmid expressing 3-OST-6 (C). Cells were fixed and then stained with Giemsa. Shown are the photographs of representative cell monolayers taken after 24 h. Arrows (in C) indicate the polykaryocyte cells.

Figure 8. Expression of 3-OST-6 mRNA in mouse tissues

A mouse MTN blot was hybridized with mouse 3-OST-6 cDNA probes labelled with [³²P]dCTP under the conditions described in the Materials and methods section. Sizes (in kb) are indicated to the left of the blot.