Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 May 17;145(4):899-910.
doi: 10.1083/jcb.145.4.899.

Sulfotransferases of two specificities function in the reconstitution of high endothelial cell ligands for L-selectin

A Bistrup  1 , S Bhakta, J K Lee, Y Y Belov, M D Gunn, F R Zuo, C C Huang, R Kannagi, S D Rosen, S Hemmerich
Affiliations

Sulfotransferases of two specificities function in the reconstitution of high endothelial cell ligands for L-selectin

A Bistrup et al. J Cell Biol. .

Abstract

L-selectin, a lectin-like receptor, mediates rolling of lymphocytes on high endothelial venules (HEVs) in secondary lymphoid organs by interacting with HEV ligands. These ligands consist of a complex of sialomucins, candidates for which are glycosylation- dependent cell adhesion molecule 1 (GlyCAM-1), CD34, and podocalyxin. The ligands must be sialylated, fucosylated, and sulfated for optimal recognition by L-selectin. Our previous structural characterization of GlyCAM-1 has demonstrated two sulfation modifications, Gal-6-sulfate and GlcNAc-6-sulfate in the context of sialyl Lewis x. We now report the cloning of a Gal-6-sulfotransferase and a GlcNAc-6-sulfotransferase, which can modify GlyCAM-1 and CD34. The Gal-6-sulfotransferase shows a wide tissue distribution. In contrast, the GlcNAc-6-sulfotransferase is highly restricted to HEVs, as revealed by Northern analysis and in situ hybridization. Expression of either enzyme in Chinese hamster ovary cells, along with CD34 and fucosyltransferase VII, results in ligand activity, as detected by binding of an L-selectin/IgM chimera. When coexpressed, the two sulfotransferases synergize to produce strongly enhanced chimera binding.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Sulfated O-linked carbohydrate chains of GlyCAM-1. Oligosaccharides bearing the 6-sulfo sLex and the sialyl 6′-sulfo Lex motif, which extend from the core 2 structure, are shown. The presence of the sialyl 6′,6-disulfo Lex motif is strongly suspected. Structures of the more complex O-linked chains of GlyCAM-1 remain to be determined.
Figure 3
Figure 3
Molecular features of human and mouse HEC-GlcNAc6ST. (A) cDNA sequence for human HEC-GlcNAc6ST and predicted protein sequences of human (h) and mouse (m) HEC-GlcNAc6STs (EMBL/GenBank/DDBJ accession nos. AF131325 and AF131236, respectively). The open reading frame is denoted by capital letters, and the predicted amino acid sequences are indicated below the nucleotide sequence. The putative transmembrane domains are underlined, and three potential N-linked glycosylation sites (for each sequence) are indicated by asterisks. (B) Alignment of regions of high conservation among human carbohydrate 6-sulfotransferases (Gal-6, GlcNAc-6, and GalNAc-6). Protein sequences were aligned using the ClustalW algorithm (Thompson et al., 1994). Black shading indicates identity at that residue among at least three of the sequences; grey shading indicates similarity among the shaded residues.
Figure 3
Figure 3
Molecular features of human and mouse HEC-GlcNAc6ST. (A) cDNA sequence for human HEC-GlcNAc6ST and predicted protein sequences of human (h) and mouse (m) HEC-GlcNAc6STs (EMBL/GenBank/DDBJ accession nos. AF131325 and AF131236, respectively). The open reading frame is denoted by capital letters, and the predicted amino acid sequences are indicated below the nucleotide sequence. The putative transmembrane domains are underlined, and three potential N-linked glycosylation sites (for each sequence) are indicated by asterisks. (B) Alignment of regions of high conservation among human carbohydrate 6-sulfotransferases (Gal-6, GlcNAc-6, and GalNAc-6). Protein sequences were aligned using the ClustalW algorithm (Thompson et al., 1994). Black shading indicates identity at that residue among at least three of the sequences; grey shading indicates similarity among the shaded residues.
Figure 2
Figure 2
Expression of HEC-GlcNAc6ST transcripts in HECs. (A) Semiquantitative RT-PCR analysis. Fragments of the HEC-GlcNAc6ST and HPRT sequences were amplified by PCR from serial dilutions of cDNA prepared from purified HECs, HUVECs, and tonsillar lymphocytes. The reaction products (456 and 300 bp, respectively) were analyzed by agarose electrophoresis and ethidium bromide staining. −RT, PCR reactions in which the template was generated by omission of RT. (B) Northern blotting. Northern blots containing poly(A)+ RNA from various human tissues (left and center) and from HECs and HUVECs (right) were probed with a 500-bp fragment from the HEC-GlcNAc6ST cDNA (top panels). The blots were stripped and reprobed with a 300-bp probe for β-actin (bottom panels).
Figure 4
Figure 4
In situ hybridization to detect HEC-GlcNAc6ST transcripts in mouse lymph node. Sections of C56BL/6 mouse lymph node were hybridized with 35S-labeled sense or antisense riboprobes based on the clone corresponding to the mouse homologue of HEC-GlcNAc6ST. Dark field micrographs of the sections are shown. Signal is seen as bright dots. (A) Hybridization with antisense probe, whole lymph node shown. The only source of signal are HEVs, seen as distinctive high-walled vessels in the cortex of the node. (B) Hybridization with sense probe of section adjacent to that in A. (C) Higher magnification view of area indicated by arrow in A. Two large HEVs are evident.
Figure 5
Figure 5
Sulfation of GlyCAM-1/IgG by sulfotransferases. COS cells were transfected with combinations of plasmids encoding GlyCAM-1/IgG, KSGal6ST, and HEC-GlcNAc6ST, as indicated. Transfected cells were cultured in the presence of [35S]sulfate, and recombinant GlyCAM-1/IgG was isolated from the CM. 1% of the captured material was analyzed by SDS-PAGE, and the remainder was subjected to hydrolysis and compositional analysis. (A) Autoradiograph (top) and Coomassie Blue staining (bottom) of SDS gel. Densitometric quantification of the Coomassie Blue–stained bands showed that each lane, except the control lane without GlyCAM-1/IgG plasmid, contained approximately equal amounts of GlyCAM-1/IgG. (B) Sulfated carbohydrates produced in GlyCAM-1 by transfection with HEC-GlcNAc6ST (▵) or KSGal6ST (•) were analyzed by Dionex chromatography after acid hydrolysis. The following standards are indicated: 1, GlcNAc-3SO3 −; 2, [35S]SO4 2−; 3, Galβ1→ 4[SO3 –→ 6]GlcNAc; 4, [SO3 –→ 6]Galβ1→ 4GlcNAc; 5, Gal-4SO3 –; 6, Gal-3SO3 –; 7, GlcNAc-6SO3 –; 8, Gal-6SO3 –.
Figure 6
Figure 6
Generation of sialyl 6-sulfo Lex by transfection with HEC-GlcNAc6ST cDNA. CHO/FTVII/C2GnT cells were transfected with a cDNA encoding HEC-GlcNAc6ST, with or without CD34 cDNA. Cells were stained with the G72 mAb to detect the presence of sialyl 6-sulfo Lex. Histogram shows G72 staining for the transfections with (red) or without (blue) CD34 cDNA, or staining of the isotype control antibody for the transfection with CD34 cDNA (black).
Figure 7
Figure 7
L-selectin reactivity conferred to CD34 by transfection with HEC-GlcNAc6ST and KSGal6ST cDNAs. CHO/FTVII/ C2GnT cells were transfected with different combinations of cDNAs encoding human CD34, HEC-GlcNAc6ST, and KSGal6ST. Cells were stained with the L-selectin/IgM chimera to detect ligand activity. (A) Two-color analysis showing CD34 expression (y-axis, staining with CD34-PE mAb) and L-selectin ligand activity (x-axis, FITC) in cells transfected with CD34 cDNA and cDNAs encoding HEC-GlcNAc6ST and KSGal6ST, alone or in combination as indicated. The horizontal bar is set such that all cells staining with the isotype-matched control for the CD34 mAb are included in the lower quadrants. The vertical bar is set to indicate the L-selectin/IgM staining of cells in which no sulfotransferase cDNA was included in the transfection mixture (bottom left panel). The fraction of positive cells (as a percentage of the total) in each quadrant is indicated. The mean fluorescence intensity (MFI) for the cells in the top right quadrants is indicated. The CD34 mAb did not interfere with staining by the L-selectin/IgM chimera. (B and C) Histograms showing L-selectin/IgM staining for the following transfections: CD34 cDNA only (blue, B and C); CD34/HEC-GlcNAc6ST/KSGal6ST cDNAs (red, B and C); CD34/HEC-GlcNAc6ST/KSGal6ST cDNAs with staining done in the presence of Mel-14 mAb (green, B); HEC-GlcNAc6/KSGal6ST cDNAs but no CD34 cDNA (green, C). The secondary staining reagent when used alone showed staining equal to that observed when Mel-14 mAb was used as an inhibitor. The isotype-matched control for Mel-14 showed no inhibitory effect.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alon R, Chen S, Puri KD, Finger EB, Springer TA. The kinetics of L-selectin tethers and the mechanics of selectin-mediated rolling. J Cell Biol. 1997;138:1169–1180. - PMC - PubMed
    1. Andrews P, Milsom D, Ford W. Migration of lymphocytes across specialized vascular endothelium. V. Production of a sulphated macromolecule by high endothelial cells in lymph nodes. J Cell Sci. 1982;57:277–292. - PubMed
    1. Baumhueter S, Dybdal N, Kyle C, Lasky LA. Global vascular expression of murine CD34, a sialomucin-like endothelial ligand for L-selectin. Blood. 1994;84:2554–2565. - PubMed
    1. Baumhueter S, Singer MS, Henzel W, Hemmerich S, Renz M, Rosen SD, Lasky LA. Binding of L-selectin to the vascular sialomucin, CD34. Science. 1993;262:436–438. - PubMed
    1. Berg EL, McEvoy LM, Berlin C, Bargatze RF, Butcher EC. L-selectin-mediated lymphocyte rolling on MAdCAM-1. Nature. 1993;366:695–698. - PubMed

Publication types

MeSH terms

Associated data