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SHORT COMMUNICATION CHST1 and CHST2 Sulfotransferases Expressed by Human Vascular Endothelial Cells: cDNA Cloning, Expression, and Chromosomal Localization Xuan Li and Thomas F. Tedder 1 Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710 Received September 10, 1998; accepted October 16, 1998
Sulfation is essential for the generation of functional vascular endothelial cell ligands for the leukocyte adhesion molecule, L-selectin. Therefore, human vascular endothelium cDNA libraries were screened to identify sulfotransferases homologous to chicken chondroitin 6-sulfotransferase (C6ST). Two sulfotransferases were identified: CHST2, a novel 530amino-acid sulfotransferase with a carboxyl-terminal region that was 45 and 43% homologous with those of human and chicken C6ST, respectively, and CHST1, which was identical to human C6ST. Northern blot analysis showed that CHST2 was broadly expressed among tissues. The CHST2 gene mapped to human chromosome 3q24 close to 3q25. Thus, this study identified two sulfotransferases expressed by vascular endothelial cells that may contribute to the generation of L-selectin ligands during inflammatory responses. © 1999 Academic Press
The selectins are adhesion molecules critical for leukocyte interactions with vascular endothelial cells during migration into tissues (13). L-selectin expressed by leukocytes mediates binding to high endothelial venules of peripheral lymph nodes and vascular endothelium during immune or inflammatory responses. L-selectin binds fucosylated O-linked carbohydrate side chains displayed in the proper context on a limited number of sulfated glycoproteins or proteoglycans (12). Sulfotransferase(s) may regulate the expression of functional L-selectin ligands since sulfation is an essential component of L-selectin HEV ligands (7, 8) and Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. AF090137 (CHST1) and AF083066 (CHST2). The CHST gene nomenclature was assigned by the HUGO/GDB Nomenclature Committees. This work was supported by NIH Grants CA54464, HL50985, and AI26872. 1 To whom correspondence should be addressed at Box 3010, Department of Immunology, Duke University Medical Center, Durham, NC 27710. Telephone: (919) 684-3578. Fax: (919) 684-8982. E-mail:
[email protected].
ligands expressed by vascular endothelial cells and leukocytes. 2 Therefore, sulfotransferase(s) expressed by human umbilical vein endothelial cells (HUVEC) and an endothelial cell line derived from HUVEC, EA.hy926 (2), were identified in this study. Golgi-associated sulfotransferases sulfonate glycoproteins, glycosaminoglycans, peptidyl tyrosine, and heparan sulfates and play important roles in intercellular communication (9). A prototype of the emerging family of Golgi membrane-bound sulfotransferases is chicken chondroitin 6-sulfotransferase (C6ST) (4). Therefore, the chicken C6ST protein sequence was used to search the translated database of expressed sequence tags (dbEST) using the BLAST program (1). Six human cDNA fragments (GenBank Accession Nos. R55609, R16177, H50963, AA243229, AA370075, and W07003) encoded small (34 – 80) amino acid stretches that were 26 –52% identical with chicken C6ST. Combinations of DNA primers based on these sequences were used to PCR amplify cDNA fragments from HUVEC and EA.hy926 cDNA libraries. One primer set amplified an ;700-bp fragment that was identical with the two recently published human sequences for keratan sulfate Gal-6-sulfotransferase (KSST) (3) and C6ST (11). Overlapping cDNA clones, termed pCHST1, isolated from the HUVEC library had nucleotide sequences identical with cDNAs encoding human KSST/ C6ST (data not shown). Another primer set, sense 59GAGGTGTTCTTTCTCTACGAGCC-39 and antisense 59-CCACGAAAGGCTTGGAGGAGG-39, amplified an ;700-bp fragment encoding a different protein also homologous to chicken C6ST. Three overlapping cDNA clones isolated from the HUVEC cDNA library generated a 2227-bp cDNA (pCHST2; Fig. 1A) that encoded a novel human gene product. The terms CHST1 and CHST2 were recommended as the official nomencla2
Tu, L., Delahunty, M. D., Ding, H., Luscinskas, F. W., and Tedder, T. F. The cutaneous lymphocyte antigen is an essential component of the L-selectin ligand induced on human vascular endothelial cells. In press.
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Genomics 55, 345–347 (1999) Article ID geno.1998.5653 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Human sulfotransferase sequences. (A) Nucleotide and deduced amino acid sequences of pCHST2 cDNA. The putative transmembrane domain is double-underlined. Regions corresponding to PCR primers used first to amplify CHST2 cDNA are underlined. Potential N-linked glycosylation sites are underlined with dots. The translation termination codon is marked by an asterisk. (B) Amino acid alignment of C6ST (chicken) with putative human CHST1 and CHST2 proteins using the BLAST program. Dashes represent identical residues, and dots indicate gaps introduced to improve the alignment. Nucleotide sequences for CHST1 and CHST2 were obtained from at least two independent cDNA clones isolated from two libraries with both strands of DNA sequenced at all positions shown.
ture for these two enzymes by the HUGO/GDB Nomenclature Committees. The pCHST2 cDNA had a 1593-bp open reading frame at the 59 end with three in-frame AUG codons (Fig. 1A). The first two methionine codons optimally fit the consensus sequence for translation initiation (10). A hydrophobic region at the amino-terminus flanked by positively charged residues (Fig. 1A) may function as a transmembrane domain for a type II protein or as a Golgi retention signal (5). The putative CHST2 protein contains four potential sites for N-glycosylation. Its carboxyl-terminal region (residues 162–530) has ;30% identity and 43% similarity with that of chicken C6ST (Fig. 1B). A polyadenylation signal sequence,
AATAAA, is 20 bp upstream from a potential poly(A) tail. The open reading frames of CHST1 and CHST2 transcripts were conserved in both HUVEC and EA.hy926 cells as determined by PCR amplification of cDNA from these libraries. By Northern blot analysis, CHST2 was expressed at the highest levels in human brain, where transcripts of ;2.8 kb (major) and ;1.8 kb (minor) were identified (Fig. 2). Similar, less intense bands were present in human heart and placenta. Bands of 7.0 kb were also seen in heart, lung, kidney, and pancreas with prolonged exposure (data not shown). It remains to be determined whether each of these bands represents tissue-specific transcript isoforms or tran-
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FIG. 2. Tissue distribution of human CHST2. A multiple-tissue Northern blot (Clontech Laboratories, Inc., Palo Alto, CA) was hybridized with a 32P-labeled CHST2 cDNA probe. Lanes 1– 8 (in order), heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. Each lane contained ;2 mg of poly(A) 1 RNA that hybridized at similar levels with b-actin probes as assessed by the manufacturer of the Northern filter. After probing was performed, the filter was washed under high stringency (0.13 SSC, 0.1% SDS, 65°C for 60 min) and exposed to an X-ray film with an intensifying screen for 24 h at 270°C.
scripts of homologous genes expressed in these tissues. The expression of CHST2 was similar to the published distribution of human CHST1 (KSST/C6ST) (3, 11), which is consistent with the distribution of chondroitin sulfate proteoglycan in the body. The chromosome location of the CHST2 gene was identified in two different homology searches. A region of pCHST2 cDNA (basepairs 1818 –2146; Fig. 1A) was 98% identical with a human sequence tag SHGC-11602 (Stanford Human Genome Center; GenBank Accession No. G14605) of the database of “Sequence Tagged Sites” (http://www.ncbi.nlm.nih.gov/dbSTS/). SHGC11602 is located on human chromosome 3 at Genomic Database locus D3S4181, which maps to 3q24 close to 3q25 (http://www.shgc.stanford.edu/Mapping/). These mapping data were confirmed by searching the UniGene collection at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/ UniGene/) for expressed sequence tags identical to pCHST2 sequences. UniGene cluster Hs.8786 contained four different expressed sequence tags identical to regions of pCHST2 (AA126782, T56257, AA243167, and N80041; expressed in uterus, spleen, and lung) that map between D3S1280 and D3S1275 on chromosome 3 region q24. Thus, CHST2 is located at 3q24, which is different from other identified human sulfotransferases (14). Human CHST1 (KSST/C6ST) has been previously mapped to 11p11.1–p11.2 (11). Chicken C6ST and human CHST1 (KSST/C6ST) have both chondroitin 6-sulfotransferase and keratan sulfate sulfotransferase activities (3, 11). Chicken C6ST also catalyzes the sulfation of sialyl lactosamine oligosaccharides (6). In addition, a sulfotransferase with an efficiency or substrate specificity different from that of CHST1 may catalyze sulfation of sLe x, a proto-
type L-selectin ligand (6). The sequence of a mouse sulfotransferase 97% identical in amino acid sequence with CHST2 was also just released by GenBank (Accession No. AB011452). This 2150-bp cDNA sequence was isolated from 7-day whole embryos and is reported to encode a protein having N-acetylglucosamine-6-Osulfotransferase activity. In our preliminary studies, CHST1 and CHST2 contribute to the generation of functional L-selectin ligands in cDNA-transfected COS cells (data not shown). Thus, the expression of CHST1 and CHST2 by human endothelial cells suggests that these sulfotransferases may be important for the transfer of sulfate to either oligosaccharides present on L-selectin ligands or other acceptor moieties expressed in vivo by HEV and at sites of inflammation. REFERENCES 1.
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