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. 2020 Aug 28;295(35):12525-12536.
doi: 10.1074/jbc.RA120.014700. Epub 2020 Jul 15.

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

Carolyn May  1 , Suena Ji  1 , Zulfeqhar A Syed  1 , Leslie Revoredo  1   2 , Earnest James Paul Daniel  2   3 , Thomas A Gerken  2   3 , Lawrence A Tabak  4 , Nadine L Samara  5 , Kelly G Ten Hagen  6
Affiliations

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

Carolyn May et al. J Biol Chem. .

Abstract

Mucin-type O-glycosylation is an essential post-translational modification required for protein secretion, extracellular matrix formation, and organ growth. O-Glycosylation is initiated by a large family of enzymes (GALNTs in mammals and PGANTs in Drosophila) that catalyze the addition of GalNAc onto the hydroxyl groups of serines or threonines in protein substrates. These enzymes contain two functional domains: a catalytic domain and a C-terminal ricin-like lectin domain comprised of three potential GalNAc recognition repeats termed α, β, and γ. The catalytic domain is responsible for binding donor and acceptor substrates and catalyzing transfer of GalNAc, whereas the lectin domain recognizes more distant extant GalNAc on previously glycosylated substrates. We previously demonstrated a novel role for the α repeat of lectin domain in influencing charged peptide preferences. Here, we further interrogate how the differentially spliced α repeat of the PGANT9A and PGANT9B O-glycosyltransferases confers distinct preferences for a variety of endogenous substrates. Through biochemical analyses and in silico modeling using preferred substrates, we find that a combination of charged residues within the α repeat and charged residues in the flexible gating loop of the catalytic domain distinctively influence the peptide substrate preferences of each splice variant. Moreover, PGANT9A and PGANT9B also display unique glycopeptide preferences. These data illustrate how changes within the noncatalytic lectin domain can alter the recognition of both peptide and glycopeptide substrates. Overall, our results elucidate a novel mechanism for modulating substrate preferences of O-glycosyltransferases via alternative splicing within specific subregions of functional domains.

Keywords: Drosophila; Galnt; O-glycosylation; PGANT; alternative splicing; lectin; lectin domain; mucin; salivary gland; secretion; splicing.

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Conflict of interest statement

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.
PGANT9A and PGANT9B glycosylate diverse mucin-based substrates. A, PGANT9A and PGANT9B are identical with the exception of the differentially spliced α repeat of the lectin domain. B, amino acid sequences of the differentially spliced α repeats are shown. Positively charged residues are highlighted blue, and negatively charged residues are highlighted red. C, peptides based on endogenous mucin substrates were used in in vitro assays to determine enzymatic activity of PGANT9A and PGANT9B. Both PGANT9A and PGANT9B were able to glycosylate a number of mucin-based peptides. D, peptides used in each reaction are shown. Each data point represents an individual assay. Error bars show S.D. Each set of assays was repeated three times.
Figure 2.
Figure 2.
Peptide preference can be altered by modifying peptide charge. Peptides and their oppositely charged versions were used for in vitro enzymatic assays. PGANT9A and PGANT9B were tested with Sgs3 and Sgs3-E (A), Sgs1 and Sgs1-E (B), Muc18B and Muc18B-K (C), and Muc11A and Muc11A-K (D). The peptides used in each reaction are shown to the right. Each data point represents an individual assay. Error bars show S.D. Each set of assays was repeated three times. Student's t test was used to calculate p values. *, p < 0.05; **, p < 0.01.
Figure 3.
Figure 3.
Position of negatively charged residue dramatically affects PGANT9B activity. A and B, peptides based on Sgs3 were used to test the effect of a negatively charged residue on the activity of PGANT9A (A) and PGANT9B (B) in vitro. Each data point represents an individual assay. C, peptides used in each reaction are shown. Error bars show S.D. Each set of assays was repeated three times. Statistical comparisons were performed between Sgs3 and each peptide variant using the Student's t test to calculate p values. *, p < 0.05; **, p < 0.01.
Figure 4.
Figure 4.
PGANT9A and PGANT9B preferred sites of addition. A and B, single-site acceptors based on the Sgs3 sequence were used to determine the preferred sites of addition by PGANT9A (A) and PGANT9B (B). C, peptides based on the preferred single-site acceptor, Sgs3-AT8, were designed to determine whether proline is important for activity. There was no activity detected for PGANT9A with these peptides. D, peptides used in each reaction are shown. Error bars show S.D. Each set of assays was repeated three times. Statistical comparisons were performed between the first peptide shown and each other variant using the Student's t test to calculate p values. **, p < 0.01.
Figure 5.
Figure 5.
In silico modeling of substrates in PGANT9A and PGANT9B. A and B, PGANT9B bound to the positively charged Sgs3 peptide (A) and bound to the Sgs3-1E5 peptide (B) containing a negative charge (E5) at the N terminus. The catalytic domain of PGANT9B is shown in green, and the lectin domain is pink. Both the catalytic loop (His437–Gly450) and the α repeat are shown as electrostatic potential surfaces, where blue indicates electropositive potential, and red indicates electronegative potential. The peptides are shown as electrostatic potential spheres, and the Lys (K5 in A) or Glu (E5 in B) at position 5 is shown as a stick. C, model of PGANT9A bound to the negatively charged Sgs3-E peptide, where the catalytic domain is shown in wheat, and the lectin domain is blue. D, a sequence analysis of the catalytic flexible loop of human and Drosophila GalNAc-Ts showing that a cluster of positive charges RKRH are conserved in the loop, corresponding to RKRS at AA 440–443 of PGANT9A and PGANT9B shown in E. E, the preference of a negative charge at the N terminus of the peptide is dictated by the positive charges in the catalytic flexible loop, highlighted as blue letters. Starred residues indicate additional positive charges in the catalytic loop of PGANT9A and PGANT9B that are not highly conserved in Drosophila and human isoforms, as shown in D.
Figure 6.
Figure 6.
PGANT9A and PGANT9B display unique preferences for charged residues N- and C-terminal to the site of glycosylation. A, sequences of peptides containing neutral, acidic, or basic residues N- and C-terminal to the potential site of glycosylation are shown. B and C, percentage of glycosylation by PGANT9A (B) and PGANTB (C) against the differently charged peptides is shown. Error bars show S.D. All assays were repeated three times. Statistical analyses are shown in Fig. S5.
Figure 7.
Figure 7.
PGANT9A and PGANT9B display unique activities on glycopeptide substrates. A, PGANT9A and PGANT9B were tested against peptide and glycopeptide substrates in in vitro enzymatic assays. B, sequences of the peptides and glycopeptides used are shown. Each data point represents an individual assay. Error bars show S.D. Each set of assays was repeated three times.
Figure 8.
Figure 8.
PGANT9A and PGANT9B display different orientation preferences for glycopeptides. A, for lectin domain binding, peptides were designed with a single C- or N-terminal GalNAc-O-Thr residue (T*), flanked by five randomized residues (magenta) containing no acceptor, and followed by 12 randomized residues (green) including the putative sites of GalNAc addition. Control peptides have Ala residue instead of GalNac-O-Thr. B, representative time-course plots for showing the net [3H]GalNAc utilization. C, Sephadex G10 chromatograms with overnight incubations demonstrating high [3H]GalNAc transfer to (glyco)peptide substrates (fractions 27–33) with minimal hydrolysis (i.e. free GalNAc, fractions 37–43). D, ratios of transfer GP(T*22)R versus GP(T*10)L (normalized to DPM/OD). For PGANT9A, n = 12 and for PGANT9B (n = 14). E, schematic drawing of orientation preferences for PGANT9A and PGANT9B.
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