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. 2009 Feb;29(4):943-52.
doi: 10.1128/MCB.02085-07. Epub 2008 Dec 15.

Core glycosylation of collagen is initiated by two beta(1-O)galactosyltransferases

Belinda Schegg  1 , Andreas J Hülsmeier, Christoph Rutschmann, Charlotte Maag, Thierry Hennet
Affiliations

Core glycosylation of collagen is initiated by two beta(1-O)galactosyltransferases

Belinda Schegg et al. Mol Cell Biol. 2009 Feb.

Abstract

Collagen is a trimer of three left-handed alpha chains representing repeats of the motif Gly-X-Y, where (hydroxy)proline and (hydroxy)lysine residues are often found at positions X and Y. Selected hydroxylysines are further modified by the addition of galactose and glucose-galactose units. Collagen glycosylation takes place in the endoplasmic reticulum before triple-helix formation and is mediated by beta(1-O)galactosyl- and alpha(1-2)glucosyltransferase enzymes. We have identified two collagen galactosyltransferases using affinity chromatography and tandem mass spectrometry protein sequencing. The two collagen beta(1-O)galactosyltransferases corresponded to the GLT25D1 and GLT25D2 proteins. Recombinant GLT25D1 and GLT25D2 enzymes showed a strong galactosyltransferase activity toward various types of collagen and toward the serum mannose-binding lectin MBL, which contains a collagen domain. Amino acid analysis of the products of GLT25D1 and GLT25D2 reactions confirmed the transfer of galactose to hydroxylysine residues. The GLT25D1 gene is constitutively expressed in human tissues, whereas the GLT25D2 gene is expressed only at low levels in the nervous system. The GLT25D1 and GLT25D2 enzymes are similar to CEECAM1, to which we could not attribute any collagen galactosyltransferase activity. The GLT25D1 and GLT25D2 genes now allow addressing of the biological significance of collagen glycosylation and the importance of this posttranslational modification in the etiology of connective tissue disorders.

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Figures

FIG. 1.
FIG. 1.
ColGalT identification by mass spectrometry. Proteins isolated by affinity chromatography were analyzed by liquid chromatography-MS. (A) Peptide fragment spectra of two peptides identifying GLT25D2. (B) Protein sequence of Gallus gallus GLT25D2. The two identifying peptides are shaded in gray, the four potential N-glycosylation sites are underlined, and the ER retrieval signal is shown in bold.
FIG. 2.
FIG. 2.
Protein alignment. The three putative human ColGalT enzymes share a high degree of sequence identity (63% between GLT25D1 and GLT25D2, 50% between GLT25D2 and CEECAM1, and 55% between GLT25D1 and CEECAM1). The proteins include the C-terminal RDEL ER retrieval motif. Black squares represent amino acids identical or similar in all three proteins; gray squares represent amino acids identical or similar in two of the proteins.
FIG. 3.
FIG. 3.
ColGalT activity toward MBL. MBL was produced in Sf9 cells coinfected with a baculovirus expressing LH3. A ColGalT activity assay was performed as described in Materials and Methods. Bars indicate the means for four assays. Error bars indicate the standard deviations.
FIG. 4.
FIG. 4.
Time course of baculovirus-mediated protein expression in Sf9 cells. ColGalT activity (A) or collagen glucosyltransferase activity (B) was measured in cells expressing GLT25D1, GLT25D2, CEECAM1, or LH3. Bovine Achilles collagen type I was used as an acceptor substrate. The activity measured in Sf9 cells infected with an empty baculovirus is shown in both panels with filled squares. Values indicate the means for four assays. Error bars indicate the standard deviations. (C) SDS-PAGE of recombinantly expressed proteins. Arrows indicate the recombinant protein bands, as confirmed by liquid chromatography-MS-mediated protein sequencing.
FIG. 5.
FIG. 5.
Determination of the apparent Km values of GLT25D1 and GLT25D2. (A) Lineweaver-Burk blot for GLT25D1 on collagen, with the calculated Michaelis-Menten constant of 13.6 g/liter. (B) Lineweaver-Burk blot for GLT25D1 on UDP-Gal, with the calculated Michaelis-Menten constant of 18.77 μM. (C) Lineweaver-Burk blot for GLT25D2 on collagen type I, with the calculated Michaelis-Menten constant of 9.8 g/liter. (D) Lineweaver-Burk blot for GLT25D2 on UDP-Gal, with the calculated Michaelis-Menten constant of 33.53 μM.
FIG. 6.
FIG. 6.
Product identification by reverse-phase HPLC. The first panel represents an amino acid standard containing the standards for GHyl and GGHyl. The second and third panels show the amino acid profiles of bovine collagen type I and type II hydrolysates, respectively. The lower two panels show the radioactive trace obtained after reaction of collagen type I with GLT25D1 and GLT25D2. [3H]Val and [14C]Tyr were used as internal amino acid standards. Amino acids are marked in single-letter code. Hyp, hydroxyproline.
FIG. 7.
FIG. 7.
Silencing of the GLT25D1 gene. (A) RT-PCR detection of GLT25D1 and GLT25D2 expression in HeLa cells. (B) mRNA GLT25D1 levels in wild-type HeLa cells (black bar) and in GLT25D1-silenced HeLa cells (KD #1 to KD #3, white bars). (C) Relative ColGalT activity in wild-type HeLa cells (black bar) and in GLT25D1-silenced HeLa cells (white bars). (D) Comparison between GLT25D1 mRNA levels and ColGalT activity in wild-type HeLa cells (set to 100%) and those in GLT25D1-silenced HeLa cells.
FIG. 8.
FIG. 8.
Tissue Northern blotting. The mRNA expression patterns of GLT25D1, GLT25D2, and CEECAM1 were analyzed in 10 human tissues (A) or in 36 human tissues and cell lines (B); a representative collection of additional tissues and cell types is shown in Fig. S2 in the supplemental material. PBL, peripheral blood leukocytes.
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