Initiation of Mammalian O-Mannosylation in Vivo Is Independent of a Consensus Sequence and Controlled by Peptide Regions within and Upstream of the α-Dystroglycan Mucin Domain

Isabelle Breloy,Tilo Schwientek,Barbara Gries,Hanieh Razawi,Marcus Macht,Christian Albers,Franz-Georg Hanisch

doi:10.1074/jbc.m802834200

Isabelle Breloy, Tilo Schwientek + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m802834200

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Abstract

To reveal insight into the initiation of mammalian O-mannosylation in vivo, recombinant glycosylation probes containing sections of human alpha-dystroglycan (hDG) were expressed in epithelial cell lines. We demonstrate that O-mannosylation within the mucin domain of hDG occurs preferentially at Thr/Ser residues that are flanked by basic amino acids. Protein O-mannosylation is independent of a consensus sequence, but strictly dependent on a peptide region located upstream of the mucin domain. This peptide region cannot be replaced by other N-terminal peptides, however, it is not sufficient to induce O-mannosylation on a structurally distinct mucin domain in hybrid constructs. The presented in vivo evidence for a more complex regulation of mammalian O-mannosylation contrasts with a recent in vitro study of O-mannosylation in human alpha-dystroglycan peptides indicating the existence of an 18-meric consensus sequence. We demonstrate in vivo that the entire region p377-417 is necessary and sufficient for O-mannosylation initiation of hDG, but not of MUC1 tandem repeats. The feature of a doubly controlled initiation process distinguishes mammalian O-mannosylation from other types of O-glycosylation, which are largely controlled by structural properties of the substrate positions and their local peptide environment.

Highlights

The posttranslational modification of proteins by O-glycosylation represents a much more complex phenomenon than initially assumed
Generation and Expression of Recombinant Glycosylation Probes—In an attempt to localize the structural elements required for O-mannosylation we followed a recombinant approach that was based on glycosylation probes corresponding to sections of human ␣-dystroglycan (Fig. 1)
Based on structural analyses of recombinant human ␣DG glycosylation probes this study provides evidence for unique mechanisms involved in the control of mammalian O-mannosylation initia

Summary

EXPERIMENTAL PROCEDURES

Materials—All chemicals were purchased from Sigma and were of the grade “pro analysi.” Exceptions are marked in the text. Enzymatic digestion of the amplified cDNA and the vector pCEP-PU with restriction endonucleases BamHI, BglII, EcoRV, and NheI (New England Biolabs, Frankfurt, Germany) was done according to the manufacturer’s protocol. The DNA of the tandem repeat domain of a MUC1 construct [36] was isolated with the restriction enzymes EcoRV (3Ј) and BamHI (5Ј) Both DNA fragments were cloned in one step into the vector pCEP-PU as described above. Masses were scanned between 100 and 450 Da. Glycopeptide Analysis by Collision-induced Dissociation(CID) Electrospray Mass Spectrometry—Prior to trypsin digestion (Promega, Mannheim, Germany) the glycoproteins were either partially deglycosylated by successive incubation with the exoglycosidases neuraminidase (Clostridium perfringens, New England Biolabs), ␤-galactosidase and ␤-N-acetylhexosaminidase (both Jack Bean, GLYKO, ProZyme, Nils, IL), or chemically desialylated in 0.1 M trifluoroacetic acid by heating for 1 h at 80 °C. The mixture was incubated for 24 h at 37 °C and stopped with 0.1% trifluoroacetic acid in water. (Glyco) Peptides were analyzed by MALDI-MS as described above

RESULTS

Peptide Glycosylation Glycan composition identity position

In vitro

DISCUSSION