The human collagen beta(1-O)galactosyltransferase, GLT25D1, is a soluble endoplasmic reticulum localized protein.
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.
Liefhebber
Jolanda Mp
JM
Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.
Punt
Simone
S
Spaan
Willy Jm
WJ
van Leeuwen
Hans C
HC
D045744:Cell Line, Tumor; D003094:Collagen / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry* / Q000378:metabolism; D005700:Galactosyltransferases / Q000032:analysis* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D037601:Mannose-Binding Lectin / Q000378:metabolism; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000378:metabolism
D037601:Mannose-Binding Lectin; D003094:Collagen; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; D005700:Galactosyltransferases; C535153:COLGALT1 protein, human
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