Representative GT41 members, HMW1C sequences, and schematics of recombinant proteins used in this study.

(A) Domain organization of three GT41 members, including the human OGT (HsOGT), the Xanthomonas campestris OGT (XcOGT), and Haemophilus influenzae HMW1C protein (HMW1C). The TPR and GT domains are indicated in blue and cyan, respectively. Based on the XcOGT structure [17], [18], domain boundaries of HsOGT were assigned (the nucleus localization signal in red). Fly and mammalian OGTs have a large insertion (in white) within the GT domain. In HMW1C, the N-terminal domain (in magenta) is different from the TPR domains in HsOGT and XcOGT. HMW1C residues 155 and 260 correspond to ApHMW1C residue 125 (limited proteolysis boundary) and XcOGT residue 203 (boundary for GT), respectively. (B) The sequence alignment of HMW1C (Hi) with its ortholog from A. pleuropneumoniae (Ap). The protease cleavage site is indicated with star. (C) Schematics of HMW1 and acceptor protein constructs. The known domain organization of HMW1 is shown: SP, the signal peptide (residues 1–68); HMW1-PP, the HMW1 pro-piece (residues 69–441) containing the secretion domain; and the mature adhesin (residues 442–1536). Several constructs representing different regions of HMW1 were generated as GST-fusion proteins to serve as acceptor proteins. Based on assessment of solubility and stability of each protein in solution, the best substrate was HMW1ct. For the substrate HMW1ct, a His-tagged version was also produced. The N-glycosylation sites, N1348, N1352, and N1366, within HMW1ct are indicated. (D) Schematics of enzyme constructs. ApHMW1C (GenBank: ABN74719.1) and its two sub-domains (P15 and P55) identified from the analysis of limited proteolysis were produced as His-tagged proteins. An analytical gel filtration profile of purified ApHMW1C (marked with star) revealed a calculated molecular weight of ∼70 kDa, consistent with a monomer. The peak positions of molecular standards are indicated as arrowheads (aldolase, 158 kDa; conalbumin, 75 kDa; and ovalbumin, 43 kDa).



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