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Comp Biochem Physiol B Biochem Mol Biol
2015 Feb 01;180:40-7. doi: 10.1016/j.cbpb.2014.10.003.
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Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis.
Voglmeir J
,
Laurent N
,
Flitsch SL
,
Oelgeschläger M
,
Wilson IB
.
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The biosynthesis of mucin-type O-linked glycans in animals is initiated by members of the large family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts), which play important roles in embryogenesis, organogenesis, adult tissue homeostasis and carcinogenesis. Until now, the mammalian forms of these enzymes have been the best characterized. However, two N-acetylgalactosaminyltransferases (xGalNAc-T6 and xGalNAc-T16) from the African clawed frog (Xenopus laevis), which are most homologous to those encoded by the human GALNT6 and GALNT16 (GALNTL1) genes, were shown to have contrasting roles in TGF-β/BMP signaling in embryogenesis. In this study we have examined these two enzymes further and show differences in their in vivo function during X. laevis embyrogenesis as evidenced by in situ hybridization and overexpression experiments. In terms of enzymatic activity, both enzymes were found to be active towards the EA2 peptide, but display differential activity towards a peptide based on the sequence of ActR-IIB, a receptor relevant to TGF-β/BMP signaling. In summary, these data demonstrate that these two enzymes from different branches of the N-acetylgalactosaminyltransferase do not only display differential substrate specificities, but also specific and distinct expression pattern and biological activities in vivo.
Fig. 1. Biological characterization of Xenopus xGalNAc-T6 and –T16. (A) Expression analysis by in situ hybridization of stage 12 (dorsal view) and stage 24 Xenopus embryos. Whereas xGalNAc-T16 is expressed in the anteriormesoderm and in the deep layer of the lateral neural plate at stage 12 and in anteriorbrain, neural crest mediolateral spinal cord and notochord at stage 24 (see also Herr et al., 2008), GalNAc-T16 is highly expressed in the ectoderm, but excluded from the neuroectoderm at stage 12 and highly expressed in the otic vesicle (ov) and cement gland (cg) at stage 24. Paraffin sections, at the indicated positions of the stage 24 and stage 12 embryos stained for xGalNAc-T6 mRNA, are shown below. (B) RT-PCR analysis of stage 20 ectodermal explants isolated from embryos microinjected four times animally at the 4–8 cell stage with 20 pg noggin mRNA, 100 pg op-1 mRNA or both. The expression of six3 and ncad indicated neuralization of the explants, xvent-2 was used as a marker for active BMP signaling, a-actin as a marker for mesoderm and odc as a loading control. (C) Typical phenotypes of untreated Xenopus embryos and embryos microinjected twice dorsal-animally at the 4–8 cell stage with 400 pg mRNA encoding xGalNAc-T6 or xGalNAc-T16. All microinjection experiments were performed at least three times (n > 25) with > 70% of the embryos displaying an reduction of head and eye structures after overexpression of GalNAc-T6 or a expansion of anterior structures, in particular of the cement gland, after GalNAc-T16 injections as described by Herr et al. (2008).
Fig. 2. Activity of frog xGalNAc-T6 and –T16 towards the EA2 and ActR-IIB peptides. A: The activities of the xGalNAc-T6 (after 30 and 60 min; 3.6 μg protein) and -T16 (after 2 and 4 h; 13.4 μg protein) were tested in vitro towards the EA2 peptide and products were identified by either RP-HPLC as evidenced by the appearance of a peak of lower retention time; the inset is of an anti-FLAG Western blot of the two enzyme preparations. B: Incubations of xGalNAc-T6 and -T16 with the ActR-IIB peptide were analysed by MALDI-TOF MS (lower panel; products with m/z 1768 and 1972); significant activity (ca. 10% conversion; m/z 1769) was observed only with xGalNAc-T16, but traces of singly- and doubly-glycosylated products were observed with xGalNAc-T6 (products of m/z 1768 and 1972 were observed with a magnified scaling factor of 500).
Fig. 3. Tandem mass spectrometric analysis of xGalNAcT products. A modified form of the EA2 peptide was employed in a MALDI-ToF-based assay and the fragmentation patterns of the substrate (A; m/z 1345), the mono- and di-glycosylated products of xGalNAc-T6 (B and C; m/z 1548 and 1751) and the sole product of xGalNAc-T16 (m/z 1548) were compared after overnight incubation. Series of putative y-fragments are annotated with amino acids (one-letter code) commencing with the C-terminal arginine residue (y1 ion of 175); a mass difference of 304 corresponds to a glycosylated threonine, whereas mass differences of − 17 may result from loss of ammonia from y-ions. The sequences of the glycosylated peptides are shown with the putatively-modified threonines indicated with asterisks.
Fig. 4. Biochemical characterization of frog xGalNAc-T6 and -T16. The following parameters were measured for both enzymes using the EA2 peptide: (A) temperature optimum, (B) cation dependence and (C) pH optimum. The conditions of time and enzyme dilution were shown for both enzymes to result in the transfer of a single GalNAc residue.
Fig. 5. A phylogeny of polypeptide-modifying N-acetylgalactosaminyltransferases. The NCBI databank was searched for frog, fish, chicken, bovine, murine and human homologues of GalNAc-T1 through to -T20; frog orthologues of the human T5, T8, T18 and T20 as well as mouse and chicken homologues of T8 were not found in this search. Highlighted are the T6 and T16 groups which are the focus of the current study.
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