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, 392 (Pt 3), 665-74

Molecular Cloning and Expression of a Human hST8Sia VI (alpha2,8-sialyltransferase) Responsible for the Synthesis of the diSia Motif on O-glycosylproteins

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Molecular Cloning and Expression of a Human hST8Sia VI (alpha2,8-sialyltransferase) Responsible for the Synthesis of the diSia Motif on O-glycosylproteins

Mélanie Teintenier-Lelièvre et al. Biochem J.

Erratum in

  • Biochem J. 2007 Feb 1;401(3):763

Abstract

Based on BLAST analysis of the human and mouse genome databases using the human CMP sialic acid; alpha2,8-sialyltransferase cDNA (hST8Sia I; EC 2.4.99.8), a putative sialyltransferase gene, was identified on human chromosome 10. The genomic organization was found to be similar to that of hST8Sia I and hST8Sia V. Transcriptional expression analysis showed that the newly identified gene was constitutively expressed at low levels in various human tissues and cell lines. We have isolated a full-length cDNA clone from the breast cancer cell line MCF-7 that encoded a type II membrane protein of 398 amino acid residues with the conserved motifs of sialyltransferases. We have established a mammary cell line (MDA-MB-231) stably transfected with the full-length hST8Sia VI and the analysis of sialylated carbohydrate structures expressed at the cell surface clearly indicated the disappearance of Neu5Acalpha2-3-sialylated structures. The transient expression of a truncated soluble form of the enzyme in either COS-7 cells or insect Sf-9 cells led to the production of an active enzyme in which substrate specificity was determined. Detailed substrate specificity analysis of the hST8Sia VI recombinant enzyme in vitro, revealed that this enzyme required the trisaccharide Neu5Acalpha2-3Galbeta1-3GalNAc (where Neu5Ac is N-acetylneuraminic acid and GalNAc is N-acetylgalactosamine) to generate diSia (disialic acid) motifs specifically on O-glycans.

Figures

Figure 1
Figure 1. Genomic organization and exon/intron junctions of the hST8Sia VI gene
(A) The hST8Sia VI gene located on chromosome 10p12.31, spans 140 kb and contains 8 exons (labelled El–E8). Black lines represent the DNA sequences identified in the human genomic databases. The entire genomic sequence of hST8Sia VI is included in the human contig: NT 008682.3. (B) Schematic representation of the hST8Sia VI mRNA. The grey boxes represent the open reading frame and the open boxes the 5′ and 3′ untranslated regions respectively. The black lines represent the mouse (m) and human (h) EST identified in the public databases. (C) The nt sequences at the intron (lowercase letters) and exon (uppercase letters) junctions are shown. Exons are numbered from the 5′ end with the initiator methionine denoted as +1.
Figure 2
Figure 2. nt and predicted amino acid sequences of hST8Sia VI
Numbering of the cDNA begins with the initiation codon. The amino acid sequence is shown in single-letter code. The putative 19 amino acid N-terminal transmembrane domain is underlined. Putative N-glycosylation sites are circled. Dashed and dotted lines underline the sialyl motifs L and S respectively. The conserved His and Glu residues in sialyl motif VS are boxed.
Figure 3
Figure 3. RT-PCR analysis of the expression of hST8Sia VI gene in various human cell lines
(A) mRNAs levels of hST8Sia VI were evaluated by RT-PCR, as described in the Experimental section, in various cell lines; lane 1, NBEC; lane 2, MCF-7; lane 3, MDA-MB-231; lane 4, T47-D; lane 5, HT-29; lane 6, Caco-2; lane 7, NCI; lane 8, Dami. (B) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as standard as previously described [50]. The sizes of the amplified fragments are indicated on the right side of the gel.
Figure 4
Figure 4. Expression of full-length human ST8Sia VI in MDA-MB-231 breast cancer cells
(A) The expression of human ST8Sia VI cDNA in MDA-MB-231 breast cancer cells was determined by RT-PCR. Lane 1, mock transfected cells; lane 2, pRc-CMV-L-hST8Sia VI transfected cells. The size of the amplified fragments are indicated on the right side. (B) hST8Sia VI enzymatic activity measured in selected cell clones. Enzymatic activity was measured on fetuin and results are expressed in pmol of transferred Neu5Ac residues per 106 cells/s. (C) Flow cytometry analysis of pRc-CMV-L-hST8Sia VI transfected cells. MDA-MB-231 expressing (pRc-CMV-L-ST8Sia VI transfected cells; dark grey peaks) or not expressing hST8Sia VI (mock transfected cells; light grey peaks), were subjected to MAA and SNA labelling. MAA revealed the presence of a terminal α3-linked sialic acid and SNA the presence of a terminal α6-linked sialic acid. Panel 1, negative control (secondary antibodies only); panel 2, MAA staining; panel 3, SNA staining.
Figure 5
Figure 5. N-deglycosylation of sialylated fetuin
[14C]Neu5Ac-labelled fetuin was produced using soluble recombinant hST8Sia VI and was subjected to PNGase F treatment for 0 min (lane 1), 30 min (lane 2), 60 min (lane 3) or 120 min (lane 4). The resulting products were separated by SDS/PAGE and detected by phosphorimaging. The molecular mass marker is indicated on the left side of the gel.
Figure 6
Figure 6. Linkage analysis of incorporated sialic acid
(A) [14C]Neu5Ac-labelled fetuin was produced using soluble recombinant hST8Sia VI and was subjected to sialidase treatment with Glyco® Sialidase S (specific for α2,3-linked sialic acid, lane 1), Glyco® Sialidase C (specific for α2,3-and α2,6-linked sialic acids, lane 2), Glyco® Sialidase A™ (specific for α2,3- α2,6 and α2,8/9-linked sialic acids, lane 3) or none (lane 4). The resulting products were separated by SDS/PAGE and detected by phosphorimaging. (B) Native fetuin (lanes 1 and 2) or α3-desialylated fetuin (lanes 3 and 4) were [14C]Neu5Ac-sialylated using soluble recombinant hST8Sia VI (lanes 2 and 4) or using mock-transfected cells conditioned medium (lanes 1 and 3). The resulting products were separated by SDS/PAGE and detected by phosphorimaging. The molecular mass marker is indicated on the left side.
Figure 7
Figure 7. HPLC/DMB analysis of α2,8-linked-[14C]-Neu5Ac on fetuin
Native fetuin was [14C]Neu5Ac-sialylated for 4 h using soluble recombinant hST8Sia VI or using mock-transfected cells conditioned medium and subjected to mild acid hydrolysis followed by DMB derivation. Oligosialylated derivatives were fractionated by HLPC on CarboPac PA-100 column according to the DP. Arrows indicate the retention time of standard di-, tri- and tetrasialo-DMB derivatives prepared from partially hydrolysed colominic acid. Inset: time course analysis of [14C]-labelled disialo-DMB derivatives synthesis. (◆) hST8Sia VI; (□) mock.

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