Revisiting the human polypeptide GalNAc-T1 and T13 paralogs

Abstract

Polypeptide GalNAc-transferases (GalNAc-Ts) constitute a family of 20 human glycosyltransferases (comprising 9 subfamilies), which initiate mucin-type O-glycosylation. The O-glycoproteome is thought to be differentially regulated via the different substrate specificities and expression patterns of each GalNAc-T isoforms. Here, we present a comprehensive in vitro analysis of the peptide substrate specificity of GalNAc-T13, showing that it essentially overlaps with the ubiquitous expressed GalNAc-T1 isoform found in the same subfamily as T13. We have also identified and partially characterized nine splice variants of GalNAc-T13, which add further complexity to the GalNAc-T family. Two variants with changes in their lectin domains were characterized by in vitro glycosylation assays, and one (Δ39Ex9) was inactive while the second one (Ex10b) had essentially unaltered activity. We used reverse transcription-polymerase chain reaction analysis of human neuroblastoma cell lines, normal brain and a small panel of neuroblastoma tumors to demonstrate that several splice variants (Ex10b, ΔEx9, ΔEx2-7 and ΔEx6/8-39bpEx9) were highly expressed in tumor cell lines compared with normal brain, although the functional implications remain to be unveiled. In summary, the GalNAc-T13 isoform is predicted to function similarly to GalNAc-T1 against peptide substrates in vivo, in contrast to a prior report, but is unique by being selectively expressed in the brain.

Exploring Regulation of Protein O-Glycosylation in Isogenic Human HEK293 Cells by Differential O-Glycoproteomics

“…We only found a few sites unique for GalNAc-T1 in contrast to T2 and T3, however, this may be related to low levels of expression of the close isoform GALNT13 (Fig. 1A), which has very similar activity (29). In our previous study in HepG2 cells we identified a considerably larger number of nonredundant GalNAc-T1 isoform-specific candidates (5), and expression of GALNT13 is not detectable by RNAseq.…”

Differential splicing of the lectin domain of an O-glycosyltransferase modulates both peptide and glycopeptide preferences

“…In fact, we and others have detected putative splice variants for other PGANT and GALNT family members (39,40). For example, splicing of the human GALNT13 alters both the length and charged residues within the g repeat of the lectin domain (39,40). This splicing event results in changes in the affinities for certain glycopeptide substrates but does not appear to alter directionality of GalNAc addition to glycopeptides (39,40).…”

“…Because lectin and catalytic subdomains lie in exonic regions of other members of this family (1), it suggests that splicing events that modify these domains may be used more widely by other PGANTs/ GALNTs. In fact, we and others have detected putative splice variants for other PGANT and GALNT family members (39,40). For example, splicing of the human GALNT13 alters both the length and charged residues within the g repeat of the lectin domain (39,40).…”

“…For example, splicing of the human GALNT13 alters both the length and charged residues within the g repeat of the lectin domain (39,40). This splicing event results in changes in the affinities for certain glycopeptide substrates but does not appear to alter directionality of GalNAc addition to glycopeptides (39,40). Whether splicing alters specific peptide preferences remains to be examined in more detail.…”

Glycans and Glycan-Binding Proteins as Regulators and Potential Targets in Leukocyte Recruitment

“…Interestingly, a recent study by May et al ( 2020 ) has shown that alternative splicing of PGANTs, the Drosophila analogues of mammalian ppGalNTs ( Table 1 ), which catalyse the addition of the glycan to serine or threonine, can alter the substrate and peptide preference of the enzyme. Even though this study investigates Drosophila PGANTs, a previous study has demonstrated the presence of splice variants in humans (Festari et al, 2017 ). Whether the splice variants of human ppGalNTs also affect the recognition of substrate in the same manner as the Drosophila splice variants and whether this impacts leukocyte recruitment remains unknown.…”