Abstract
The sialyl Lewis x (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)Glc-NAc) determinants serve as ligands in the selectin-mediated adhesion of leukocytes to activated endothelium or platelets. In our efforts to identify glycosyltransferases involved in the biosynthesis of those ligands, we achieved expression cloning of a novel human alpha 1,3-fucosyltransferase termed Fuc-TVII from a THP-1 cDNA library by enrichment of the Namalwa cells highly expressing that determinant with a fluorescence-activated cell sorter. Expression of the COOH-terminal catalytic domain of Fuc-TVII showed an alpha 1,3-fucosyltransferase activity for a type II oligosaccharide with a terminal alpha 2,3-linked sialic acid among various acceptors, consistent with that in vivo acceptor specificity. Alignment of the primary sequences of five alpha 1,3-fucosyltransferases and assignment of the chromosomal location of Fuc-TVII gene, together with that acceptor specificity, indicate that Fuc-TVII consists of a unique class of the alpha 1,3-fucosyltransferase family. Determination of the expression levels of these alpha 1,3-fucosyltransferases in various cells revealed that both Fuc-TVII and a myeloid fucosyltransferase Fuc-TIV were significantly expressed in myeloid lineage cells. Fuc-TVII-transfected Namalwa cells exhibited significant binding to E-selectin in contrast to little binding of the Fuc-TIV-transfected cells. These results suggest that Fuc-TVII may participate in the biosynthesis of the selectin ligands.
Highlights
Expression Cloningof a Novel al,3=FucosyltransferaseThat Is Involved in Biosynthesis of the Sialyl Lewisx Carbohydrate Determinants in Leukocytes*
NAc) determinants serve as ligands in the selectin-me- 3(Fuccul-4)GlcNAc)determinant have evoked considerable indiated adhesion of leukocytes to activated endothelium terest because both were demonstrated to serveas ligands for or platelets.In our efforts to identifyglycosyltrans- the threeknown selectins (E,P,and L-selectins) (2-ll), which ferases involved in thebiosynthesis of those ligands,we are cell adhesion molecules involved in the recruitmentof leuachieved expression cloningof a novel human a1,3-fu- kocytes into lymphoid tissues and sites of inflammation (12, cosyltransferase termed Fuc-TVII from a THP-1cDNA 13).these determinants and their related struclibrary by enrichment of the Namalwa cells highly ex- tures have been reported to be associatedwithcancer pressing that determinant with a fluorescence-activated cell sorter
Expression Cloning of cDNA Directing Biosynthesis of the sLe' Determinants-% search novel Fuc-Ts involved in thebiosynthesis of the selectin carbohydrate ligandsw, e employed the expression cloning approach using lectin resistance selection, described previously [18],and achieved the isolation of a FucTVI clone from the SW1116 cDNA library by selection with the cytostatic M.amurensis lectin I
Summary
Lewis x Biosynthesis the participationof yet unknown al,3-Fuc-T(s), which may re- of sorting on the flow cytometer. We report the expression cloning of a novel human a1,3-Fuc-T, designated Fuc-TVII, which directs thebiosynthesis of the sLe' determinant in the human Burkittlymthese cells as described [40]. Individual plasmids were examined for their ability to increase thelevel of sLe" antigens in Namalwa KJM-1 cells to select a Fuc-TVII-expressing clone named pAMo-FT7. Expression Cloning of cDNAs Encoding Fuc-TIII and Fuc-TVI-A Fuc-TIII-expressing plasmid pAMo-FT3 was selected from the SW1116 phoma cell line Namalwa. A Fuc-TVI-expressing vector pAMo-FT6 was obtained from that both Fuc-TIV and Fuc-TVII were significantly expressed in myeloid lineage cells. Either Fuc-T was found t o form the sLe" antigens at the cell surface, when expressed in Namalwa the SW1116 cDNA library by the cloning approach with resistance selection against 10pg/ml Maacia amurensis lectinI, obtained fromVector Laboratories Inc., as described previously [18]. &GCTTCACCGCTCGAACCAGCTGGC-3' (synthetic Asp7'$ site underlined), digested with HindIII and Asp7i8 and cloned between the HindIII and Asp718sites ofpAMo [18], which generated a Fuc-TIV-
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