Abstract
N-Glycosylation plays a fundamental role in many biological processes. Human diamine oxidase (hDAO), required for histamine catabolism, has multiple N-glycosylation sites, but their roles, for example in DAO secretion, are unclear. We recently reported that the N-glycosylation sites Asn-168, Asn-538, and Asn-745 in recombinant hDAO (rhDAO) carry complex-type glycans, whereas Asn-110 carries only mammalian-atypical oligomannosidic glycans. Here, we show that Asn-110 in native hDAO from amniotic fluid and Caco-2 cells, DAO from porcine kidneys, and rhDAO produced in two different HEK293 cell lines is also consistently occupied by oligomannosidic glycans. Glycans at Asn-168 were predominantly sialylated with bi- to tetra-antennary branches, and Asn-538 and Asn-745 had similar complex-type glycans with some tissue- and cell line-specific variations. The related copper-containing amine oxidase human vascular adhesion protein-1 also exclusively displayed high-mannose glycosylation at Asn-137. X-ray structures revealed that the residues adjacent to Asn-110 and Asn-137 form a highly conserved hydrophobic cleft interacting with the core trisaccharide. Asn-110 replacement with Gln completely abrogated rhDAO secretion and caused retention in the endoplasmic reticulum. Mutations of Asn-168, Asn-538, and Asn-745 reduced rhDAO secretion by 13, 71, and 32%, respectively. Asn-538/745 double and Asn-168/538/745 triple substitutions reduced rhDAO secretion by 85 and 94%. Because of their locations in the DAO structure, Asn-538 and Asn-745 glycosylations might be important for efficient DAO dimer formation. These functional results are reflected in the high evolutionary conservation of all four glycosylation sites. Human DAO is abundant only in the gastrointestinal tract, kidney, and placenta, and glycosylation seems essential for reaching high enzyme expression levels in these tissues.
Highlights
From the Departments of ‡Biotechnology, ¶Chemistry, and **Food Science and Technology, University of Natural Resources and
We show that Asn-110 in native Human diamine oxidase (hDAO) from amniotic fluid and Caco-2 cells, DAO from porcine kidneys, and recombinant hDAO (rhDAO) produced in two different HEK293 cell lines is consistently occupied by oligomannosidic glycans
The N-glycosylation profiles of native DAO purified from human amniotic fluid, human Caco-2 cells, and re-purified from commercially available porcine kidney rhDAO expressed in and purified from CHO-K1, HEK293, and HEK293-GlycoDelete cells are shown in Fig. 1 and Figs
Summary
The N-glycosylation profiles of native DAO purified from human amniotic fluid, human Caco-2 cells, and re-purified from commercially available porcine kidney rhDAO expressed in and purified from CHO-K1, HEK293, and HEK293-GlycoDelete cells are shown in Fig. 1 and Figs. The percentage distribution of the terminal carbohydrate residues and of the antennarity among the four glycosylation sites of all samples except porcine kidney DAO (pkDAO) is presented in Table 1 and Table S1, respectively. 10% Man, about 50% Gal, 27% Neu5Ac, and 20% GlcNAc terminal carbohydrate residues (Table 1) This terminal glycan distribution between Asn-538 and Asn-745 is statistically not different between the two sites (p value ⫽ 0.41; Table S2). The antennarity is very similar between the two glycan sites in our samples, with only native hDAO from amniotic fluid showing tri- and tetra-antennary branches (Table S1). In the pkDAO sample, 97% terminal Gal residues were found at Asn-541, the equivalent site to Asn-538 in human DAO. Percent distribution of terminal carbohydrate residues among the four glycosylation sites from recombinant (CHO-K1 batches 1–3; HEK293, and HEK-GD) or native (Amniotic fluid and Caco-2) human. The low sialic acid content in the pkDAO might be an artifact from the commercial isolation procedure
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