Lectin domains of polypeptide GalNAc transferases exhibit glycopeptide binding specificity

Abstract

UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (GalNAc-Ts) constitute a family of up to 20 transferases that initiate mucin-type O-glycosylation. The transferases are structurally composed of catalytic and lectin domains. Two modes have been identified for the selection of glycosylation sites by GalNAc-Ts: confined sequence recognition by the catalytic domain alone, and concerted recognition of acceptor sites and adjacent GalNAc-glycosylated sites by the catalytic and lectin domains, respectively. Thus far, only the catalytic domain has been shown to have peptide sequence specificity, whereas the primary function of the lectin domain is to increase affinity to previously glycosylated substrates. Whether the lectin domain also has peptide sequence selectivity has remained unclear. Using a glycopeptide array with a library of synthetic and recombinant glycopeptides based on sequences of mucins MUC1, MUC2, MUC4, MUC5AC, MUC6, and MUC7 as well as a random glycopeptide bead library, we examined the binding properties of four different lectin domains. The lectin domains of GalNAc-T1, -T2, -T3, and -T4 bound different subsets of small glycopeptides. These results indicate an additional level of complexity in the initiation step of O-glycosylation by GalNAc-Ts.

FIGURE 1.

Binding of GalNAc transferase lectins to mucin GalNAc glycopeptides. A, schematic depiction of GalNAc transferases. All GalNAc-Ts are single pass type II transmembrane proteins with catalytic and lectin domains. The localization of mutations in GalNAc-T2^458H, GalNAc-T2^224H, GalNAc-T3^519H, GalNAc-T4^459H, and GalNAc-T4lec^459H is shown by arrows. B, fluorescent image of a mucin glycoprotein array probed with GalNAc-T1, -T2, -T3, and -T4. Quantification of binding with the relative fluorescent units is shown on the x axis. C, GalNAc-T1, -T2, -T3, and -T4 binding to partially glycosylated mucin fragments MUC4 and MUC5AC on the array by individual GalNAc-Ts. Differentially glycosylated mucins were used as baits to probe GalNAc transferase binding.

FIGURE 2.

Influence of pH on GalNAc-T binding to MUC1 Tn glycopeptides. Binding of GalNAc-T1, -T2, -T3, and -T4 to GalNAc-MUC1 at pH 5.7–8.0. Binding is expressed as the mean relative fluorescent activity of MUC1 GalNAc-glycopeptides.

FIGURE 3.

Differential binding of GalNAc transferase lectin domains to GalNAc glycopeptides. Binding of GalNAc-T1, -T2, -T3, -T4, and HPA to the MUC4 and IgA glycopeptide array. The bar graph represents the relative fluorescent unit of the binding of each GalNAc-T. Peptide sequences are listed under supplemental Table S1. HPA, helix pomatia agglutinin.

FIGURE 4.

GalNAc-T1 selectively binds and mediates followup glycosylation of GalNAc-MUC4.1. A, binding of GalNAc-T1, -T2, -T3, and -T4 to MUC4.1, GalNAcⁿ = ^1-pos12-MUC4.1 (GalNAc incorporated at Thr-12 by GalNAc-T2, see Fig. 5 for characterization), and GalNAc-glycosylated MUC5AC (recMUC5AC Tn). B, glycosylation of GalNAcⁿ = ^1-pos12-MUC4.1 by GalNAc-T1, -T2, -T3, and -T4. C, glycosylation of GalNAcⁿ = ^1-pos12-MUC4.1 by GalNAc-T1 for 1 and 4 h in the presence of 250 mm GalNAc (upper panels) or GlcNAc (lower panels) evaluated by MALDI-TOF MS. Number of GalNAcs incorporated is indicated. Error bars indicate standard deviation of four replicates.

FIGURE 5.

Glycosylation of the MUC4.1 peptide by GalNAc-T2, followed by GalNAc-T1. Characterization of MUC4.1 products using ESI-LTQ-Orbitrap MS and ETD-MS/MS. A, MS of the MUC4.1 + 1Tn product formed by GalNAc-T2; B, MS of MUC4.1 + 2Tn product formed by subsequent action of GalNAc-T1; C, ETD-MS/MS of [M + K⁺3H]⁴⁺ precursor ion (m/z 496.4691, z = 4, in panel A); D, ETD-MS/MS of [M + K⁺3H]⁴⁺ precursor ion (m/z 547.2398, z = 4, in panel B). Inset in C, expanded region, m/z 991–995 from ETD-MS/MS of the [M + K⁺2H]³⁺ precursor ion (m/z 661.6241, z = 3, in panel A) of GalNAc-T2, showing a well resolved c₉ ion at a m/z consistent with nonglycosylation of Thr-9. The fragmentation patterns in C and D are consistent with glycosylation of Thr-10, and Thr-9 and Thr-10, respectively, with HexNAc residues (□); i.e. abundant c₇⁺, c₈⁺, c₉⁺, c₁₁⁺, z^•_9⁺, and related ions (z^•_n ± 1 u, c_n ± 1 u and/or corresponding doubly charged species) were detected at m/z values calculated for HexNAc at these sites. Corresponding fragments consistent with glycosylation elsewhere were not detected.

FIGURE 6.

Evaluation of GalNAc-T2 binding to recombinant MUC2. Left panel, binding of GalNAc-T2 and GalNAc-T2^458H to nonglycosylated recombinant MUC2. Binding assay included UDP and MnCl₂ to allow binding through the catalytic domain. Right panel, binding of GalNAc-T2 and GalNAc-T2^458H to MUC2 Tn. Error bars indicate standard deviation of four replicates.