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. 2010 May 14:11:33.
doi: 10.1186/1471-2121-11-33.

The human collagen beta(1-O)galactosyltransferase, GLT25D1, is a soluble endoplasmic reticulum localized protein

Jolanda Mp Liefhebber  1 , Simone Punt, Willy Jm Spaan, Hans C van Leeuwen
Affiliations

The human collagen beta(1-O)galactosyltransferase, GLT25D1, is a soluble endoplasmic reticulum localized protein

Jolanda Mp Liefhebber et al. BMC Cell Biol. .

Abstract

Background: Glycosyl transferases transfer glycosyl groups onto their substrate. Localization partially defines their function. Glycosyl transferase 25 domain 1 (GLT25D1) was recently shown to have galactosyltransferase activity towards collagens and another well known substrate, mannose binding lectin (MBL). To gain more insight in the role of galactosylation of lysines in the Gly-X-Lys repeats of collagenous proteins, we investigated the subcellular localization of GLT25D1.

Results: Immunofluorescence analysis of GLT25D1 expressed in the human hepatoma cell line (Huh7), revealed a perinuclear lattice like staining, resembling localization to the endoplasmic reticulum (ER). Possible targeting signals, an N-terminal signal sequence and a C-terminal ER-retention signal, were identified using prediction programs. These signals were then investigated by constructing a series of epitope-tagged forms of GLT25D1 that were analyzed by immunofluorescence and western blotting. In agreement with the predictions our results show that GLT25D1 is directed to the ER lumen as a soluble protein and retained there. Moreover, using two endoglycosidase enzymes EndoH and EndoF, we demonstrate that the putative bi-functional glycosyl transferase itself is a glycoprotein. Additionally we examined co-localization of GLT25D1 with MBL and lysyl hydroxylase 3 (LH3, PLOD3), which is a protein able to catalyze hydroxylation of lysine residues before they can be glycosylated. We demonstrate overlapping localization patterns of GLT25D1, MBL and LH3.

Conclusions: Taken together our data indicate that galactosylation of collagenous proteins by the soluble GLT25D1 occurs in the early secretory pathway.

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Figures

Figure 1
Figure 1
Subcellular localization of Glycosyltransferase 25 domain 1 protein. Huh7 cells were transfected with full length Glycosyltransferase 25 domain 1 containing an internal Myc-tag (GLT25D1) (See for more details Methods). After 24 h of expression, cells were fixed and subjected to immunofluorescence analysis. Cells were double labeled for GLT25D1 by anti-Myc (shown in red) and for the cellular markers (shown in green). ER is visualized by anti-protein disulfide isomerase (PDI) (top panels), ER-Golgi intermediate compartment by anti-ERGIC53 (middle panels) or Golgi by anti-Giantin (bottom panels). Yellow observed in the overlay indicates overlap of red and green signal. Pearsons correlation of PDI and GLT25D1 is 0.58, ERGIC53 and GLT25D1 is 0.13, and Giantin and GLT25D1 is 0.04.
Figure 2
Figure 2
Schematic representation of Glycosyltransferase 25 domain 1, including various predicted motifs. Top: Homology based structural models of the tandem glycosyltransferase domains. Bottom: Linear representation of GLT25D1 with possible signal sequence, ER retention signal and N-linked glycosylation sites indicated. Verification and discussion of these predictions are described in this article.
Figure 3
Figure 3
Characterization of signals in Glycosyl transferase 25 domain 1, suggesting ER residence. A) Summary of GLT25D1 expression constructs. HA indicates Hemagglutinin epitope tag; Myc refers to Myc epitope tag; FL denotes a full-length protein; RDEL represents carboxy-terminal Arg-Asp-Glu-Leu sequence; SS stands for signal sequence and Δ is followed by deleted part of the protein. B) Co-transfected Huh7 cells with constructs GLT25D1 FL-HA and GLT25D1 ΔSS-Myc were subjected to immunofluorescence analysis. Signal of FL-HA is shown in green and of ΔSS-Myc in red. C) The constructs 1 to 9 shown in A were transfected into Huh7 cells. The total cell lysates (Total) and the culture medium (Medium) from these cells were analyzed by SDS-PAGE and western blot. Using anti-bodies against the N- and C-terminal tags of the constructs, anti-HA and anti-Myc, the proteins were detected.
Figure 4
Figure 4
N-linked glycosylation of Glycosyl transferase 25 domain 1. A) Full length GLT25D1 (FL) or GLT25D1 with the signal sequence deleted (ΔSS) constructs were transfected into Huh7 cells. Both constructs were carboxy terminal tagged with MycHis6, to purify proteins using Co2+-beads and to detect in western blotting. Cell lysates are shown in the first two lanes after separation on SDS-PAGE and western blotting using an antibody against Myc. The purified proteins were subjected to either none (-) or Endoglycosidase H (EndoH) or F (EndoF) treatment (See for more details Methods). The results for FL protein are shown in lanes 3 to 5 and for ΔSS protein in lanes 6 to 8. B) Partial de-glycosylation treatment of GLT25D1 full length (FL).
Figure 5
Figure 5
Co-localization of Glycosyl transferase 25 domain 1 with Mannose binding lectin and Lysyl hydroxylase 3. A) Huh7 cells were co-transfected with Mannose binding lectin fused to GFP (MBL-GFP) and full length GLT25D1 tagged with Myc internally (GLT25D1), and analyzed by immunofluorescence after 24 h of expression. MBL was detected directly due to GFP and is shown in green. GLT25D1 and lysyl hydroxylase 3 (LH3) were visualized using antibodies against Myc or LH3 and are presented in red and blue, respectively. When red and green signals overlap this is observed as yellow, red and blue as purple and red, blue and green as white. Pearson correlation of GLT25D1 and MBL is 0.53, GLT25D1 and LH3 is 0.42, and LH3 and MBL is 0.45. B) A profile plot with signal intensities from MBL (green), GLT25D1 (red) and LH3 (blue) along the line drawn in the overlay on the left.
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