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. 2012 May;60(5):397-407.
doi: 10.1369/0022155412437613. Epub 2012 Jan 19.

Expression of long-form N-acetylglucosamine-6-O-sulfotransferase 1 in human high endothelial venules

Maiko Fujiwara  1 , Motohiro Kobayashi, Hitomi Hoshino, Kenji Uchimura, Tsutomu Nakada, Junya Masumoto, Yasuhiro Sakai, Minoru Fukuda, Jun Nakayama
Affiliations

Expression of long-form N-acetylglucosamine-6-O-sulfotransferase 1 in human high endothelial venules

Maiko Fujiwara et al. J Histochem Cytochem. 2012 May.

Abstract

Two members of the N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) family, GlcNAc6ST-1 and GlcNAc6ST-2, function in the biosynthesis of 6-sulfo sialyl Lewis X-capped glycoproteins expressed on high endothelial venules (HEVs) in secondary lymphoid organs. Thus, both enzymes play a critical role in L-selectin-expressing lymphocyte homing. Human GlcNAc6ST-1 is encoded by a 1593-bp open reading frame exhibiting two 5' in-frame methionine codons spaced 141 bp apart. Both resemble the consensus sequence for translation initiation. Thus, it has been hypothesized that both long and short forms of GlcNAc6ST-1 may be present, although endogenous expression of either form has not been confirmed in humans. Here, the authors developed an antibody recognizing amino acid residues between the first two human GlcNAc6ST-1 methionines. This antibody specifically recognizes the long form of the enzyme, a finding validated by Western blot analysis and immunofluorescence cytochemistry of HeLa cells misexpressing long and/or short forms of human GlcNAc6ST-1. Using this antibody, the authors carried out immunofluorescence histochemistry of human lymph node tissue sections and found endogenous expression of the long form of the enzyme in human tissue, predominantly in the trans-Golgi network of endothelial cells that form HEVs.

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Conflict of interest statement

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Nucleotide and deduced amino acid sequences of the N-terminal region of human GlcNAc6ST-1 (long form). The first two methionines are boxed, and the sequence encoding the putative transmembrane domain is doubly underlined. The sequence of the antigenic peptide used to produce anti-GlcNAc6ST-1-N antibody is underlined in bold.
Figure 2.
Figure 2.
Western blot analysis of long and short forms of human GlcNAc6ST-1. The membrane fraction of HeLa cells transiently transfected with pcDNA1 (mock), pcDNA1-GlcNAc6ST-1 M#1-FLAG, pcDNA1-GlcNAc6ST-1 M#2-FLAG, or pcDNA1-GlcNAc6ST-2, with or without PNGase F digestion, was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and probed with anti-FLAG (left panel) and anti-GlcNAc6ST-1-N (right panel) antibodies. Molecular weights are indicated to the left of each blot.
Figure 3.
Figure 3.
Specificity of anti-GlcNAc6ST-1-N antibody as assessed by immunofluorescence cytochemistry. HeLa cells were transiently transfected with expression vectors harboring cDNA encoding long (M#1) and short (M#2) forms of human GlcNAc6ST-1 with or without a FLAG epitope tag, as well as GlcNAc6ST-2 and mock (pcDNA1) as controls. Cells were doubly immunostained with anti-FLAG (green) and anti-GlcNAc6ST-1-N (red). Yellow signals in merged images indicate colocalization of the two antigens. Bar = 100 µm.
Figure 4.
Figure 4.
Subcellular localization of long and short forms of human GlcNAc6ST-1. (A) Schematic representation of long-form GlcNAc6ST-1 and short-form GlcNAc6ST-1 with a FLAG epitope tag. The antibody-binding site for anti-GlcNAc6ST-1-N (green) and anti-FLAG (red) is also indicated. LFS, long form–specific site. (B) HeLa cells doubly transfected with pcDNA1-GlcNAc6ST-1 M#1 and pcDNA1-GlcNAc6ST-1 M#2-FLAG were subjected to dual immunofluorescence staining for anti-GlcNAc6ST-1-N (green) and anti-FLAG (red). Yellow signals in merged images indicate colocalization of the two antigens. Bar = 50 µm.
Figure 5.
Figure 5.
(A) Cell enzyme-linked immunosorbent assay (ELISA) showing intracellular GlcNAc-6-O-sulfation activity of GlcNAc6ST-1 M#1 and GlcNAc6ST-1 M#2. HeLa cells were transiently transfected with pcDNA1-GlcNAc6ST-1 M#1 and pcDNA1-GlcNAc6ST-1 M#2, as well as pcDNA1-GlcNAc6ST-2 and pcDNA1 (mock) as controls, and subjected to cell ELISA for S2 monoclonal antibody recognizing 6-sulfo sialyl LacNAc on N- and O-glycans. Data are expressed as means ± SD (n = 8 for each group). NS, not significant. (B) Intracellular expression levels of long (left panel) and short (right panel) forms of GlcNAc6ST-1 protein with a C-terminal FLAG epitope tag (gray histograms). Cells were stained for FLAG and subjected to fluorescence-activated cell sorting analysis. Open histograms represent negative control resulting from omitting the primary antibody. The x- and y-axes indicate fluorescence intensity and number of events, respectively. (C) Semi-quantitative RT-PCR showing mRNA expression levels of HeLa cells transfected with long (M#1) and short (M#2) forms of GlcNAc6ST-1 cDNA. Each RNA sample was treated with (+) or without (–) reverse transcriptase (RT). Cont., control amplification using distilled water (–) and plasmid harboring the target cDNA (+). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 6.
Figure 6.
Endogenous expression of long-form GlcNAc6ST-1 protein as revealed by immunofluorescence histochemistry of human lymph node tissue sections. (A) Double immunofluorescence staining with MECA-79 (green) and anti-GlcN Ac6ST-1-N (red). Lower panels are enlarged images of the upper panels. Bar = 100 µm for upper panels and 25 µm for lower panels. (B) A competitive inhibition assay on tissue sections for anti-GlcNAc6ST-1-N binding in which antibody is preincubated at 4C overnight with 5 µg/ml of synthetic peptide used for immunization. Anti-GlcNAc6ST-1 staining on high endothelial venules (left panel) was abolished by this procedure (right panel). Bar = 100 µm. (C) Double immunofluorescence staining with anti-GlcNAc6ST-1-N (green) and one of the following Golgi markers (red): cis-Golgi marker GM130 (upper panels), medial-to-trans-Golgi marker GS27 (middle panels), and a trans-Golgi network marker to plasma membrane protein Rab8 (lower panels). Bar = 25 µm.
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