Direct Mapping of Additional Modifications on Phosphorylated O-glycans of α-Dystroglycan by Mass Spectrometry Analysis in Conjunction with Knocking Out of Causative Genes for Dystroglycanopathy.
Dystroglycanopathy is a major class of congenital muscular dystrophy caused by a deficiency of functional glycans on α-dystroglycan (αDG) with laminin-binding activity. Recent advances have led to identification of several causative gene products of dystroglycanopathy and characterization of their in vitro enzymatic activities. However, the in vivo functional roles remain equivocal for enzymes such as ISPD, FKTN, FKRP, and TMEM5 that are supposed to be involved in post-phosphoryl modifications linking the GalNAc-β3-GlcNAc-β4-Man-6-phosphate core and the outer laminin-binding glycans. Herein, by direct nano-LC-MS2/MS3analysis of tryptic glycopeptides derived from a truncated recombinant αDG expressed in the wild-type and a panel of mutated cells deficient in one of these enzymes, we sought to define the full extent of variable modifications on this phosphorylated core O-glycan at the functional Thr317/Thr319sites. We showed that the most abundant glycoforms carried a phosphorylated core at each of the two sites, with and without a single ribitol phosphate (RboP) extending from terminal HexNAc. At much lower signal intensity, a novel substituent tentatively assigned as glycerol phosphate (GroP) was additionally detected. As expected, tandem RboP extended with a GlcA-Xyl unit was only identified in wild type, whereas knocking out of either ISPD or FKTN prevented formation of RboP. In the absence of FKRP, glycoforms with single but not tandem RboP accumulated, consistent with the suggested role of this enzyme in transferring the second RboP. Intriguingly, the single GroP modification also required functional FKTN whereas absence of TMEM5 significantly hindered only the addition of RboP. Our findings thus revealed additional levels of complexity associated with the core structures, suggesting functional interplay among these enzymes through their interactions. The simplified analytical workflow developed here should facilitate rapid mapping across a wider range of cell types to gain better insights into its physiological relevance.
From the ‡Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.
Hirokazu
0000-0001-9296-0225
H
Yagi
§Institute of Biological Chemistry, Academia Sinica, 128, Academia Road Sec. 2, Nankang, Taipei 115, Taiwan.
Chu-Wei
CW
Kuo
From the ‡Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.
Takayuki
T
Obayashi
¶Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama Myodaiji, Okazaki 444-8787, Japan.
Satoshi
S
Ninagawa
§Institute of Biological Chemistry, Academia Sinica, 128, Academia Road Sec. 2, Nankang, Taipei 115, Taiwan; kkato@phar.nagoya-cu.ac.jp.
Kay-Hooi
KH
Khoo
From the ‡Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan; kkato@phar.nagoya-cu.ac.jp.|¶Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama Myodaiji, Okazaki 444-8787, Japan.
Koichi
K
Kato
D049030:Dystroglycans / Q000737:chemistry*; D055786:Gene Knockout Techniques; D020022:Genetic Predisposition to Disease; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009713:Nucleotidyltransferases / Q000235:genetics*; D010430:Pentosyltransferases; D010766:Phosphorylation; D011506:Proteins / Q000235:genetics; D058494:Walker-Warburg Syndrome / Q000235:genetics*
C113587:FKTN protein, human; D008565:Membrane Proteins; D011506:Proteins; D049030:Dystroglycans; C443107:FKRP protein, human; D010430:Pentosyltransferases; C578283:RXYLT1 protein, human; D009713:Nucleotidyltransferases; C573136:CRPPA protein, human