Glycomic survey mapping of zebrafish identifies unique sialylation pattern

Abstract

Functional genomics and proteomics studies of the developmental glycobiology of zebrafish are greatly hampered by the current lack of knowledge on its glycosylation profile. To furnish the requisite structural basis for a more insightful functional delineation and genetic manipulation, we have initiated a survey mapping of the possible expression of stage-specific glycoconjugates in zebrafish. High-sensitivity mass spectrometry (MS) analysis in conjunction with the usual array of enzymatic and chemical derivatization was employed as the principal method for rapid differential mapping of the glycolipids and sequentially liberated N- and O-glycans from the total extracts. We demonstrated that all developmental stages of the zebrafish under investigation, from fertilized eggs to hatched embryos, synthesize oligomannosyl types of N-glycans, as well as complex types with additionally beta4-galactosylated, Neu5Ac/Neu5Gc monosialylated Lewis x termini. A combination of collision-induced dissociation (CID)-MS/MS and nuclear magnetic resonance (NMR) analyses led to the identification of an abundant and unusual mucin-type O-glycosylation, based on a novel sequence Fucalpha1-3GalNAcbeta1-4(Neu5Ac/Neu5Gcalpha2-3)Galbeta1-3GalNAc. This core structure may be further oligosialylated, but exclusively in the earlier development stages. Similarly, MS and MS/MS analyses of the extracted glycolipid fraction revealed the presence of a heterogeneous family of oligosialylated lactosylceramide compounds. In contrast to the O-glycans, these glycolipids only appear in the later development stages, suggesting a complex pattern of regulation for sialyltransferase activities during zebrafish embryogenesis.

Molecular Cloning of Pigeon UDP-galactose:β-d-Galactoside α1,4-Galactosyltransferase and UDP-galactose:β-d-Galactoside β1,4-Galactosyltransferase, Two Novel Enzymes Catalyzing the Formation of Galα1–4Galβ1–4Galβ1–4GlcNAc Sequence

“…Since the presence of Gal␤1-4Gal on N-glycans from zebrafish was previously reported (28,29), the proteins containing DUF23 are candidates for ␤4GalT(Gal) in zebrafish. Gal␤1-4Gal on N-glycans was also reported to exist in medaka (30 -32) and dace (33,34).…”

A ratiometric lectin microarray approach to analysis of the dynamic mammalian glycome

“…Recent efforts toward decoding the glycome have focused on the use of mass spectrometry (4)(5)(6)(7)(8)(9). Although great strides have been made in the use of this technique, mass spectrometry has difficulty in discriminating between the many structural isomers presented by glycans.…”

Glycomics and Proteomics Analyses of Mouse Uterine Luminal Fluid Revealed a Predominance of Lewis Y and X Epitopes on Specific Protein Carriers

“…GC-MS linkage analysis on partially methylated alditol acetates prepared thereof was performed as described previously (28). For MALDI-TOF MS glycan profiling, the permethyl derivatives in acetonitrile were mixed 1:1 with 2,5-dihydroxybenzoic acid matrix (10 mg/ml in acetonitrile), spotted on the target plate, air-dried, and recrystallized on plate with acetonitrile.…”