Comparison of sialylated and fucosylated N-glycans attached to Asn 6 and Asn 41 with different roles in hyaluronan and proteoglycan link protein 1 (HAPLN1)

Abstract

Hyaluronan and proteoglycan link protein 1 (HAPLN1) is an extracellular matrix protein stabilizing interactions between hyaluronan and proteoglycan. Although HAPLN1 is being investigated for various biological roles, its N-glycosylation is poorly understood. In this study, the structure of N-glycopeptides of trypsin-treated recombinant human HAPLN1 (rhHAPLN1) expressed from CHO cells were identified by nano-liquid chromatography-tandem mass spectrometry. A total of 66 N-glycopeptides were obtained, including 16 and 12 N-glycans at sites Asn 6 (located in the N-terminal region) and Asn 41 (located in the Ig-like domain, which interacts with proteoglycan), respectively. The quantities (%) of each N-glycan relative to the totals (100 %) at each site were calculated. Tri- and tetra-sialylation (to resist proteolysis and extend half-life) were more abundant at Asn 6, and di- (core- and terminal-) fucosylation (to increase binding affinity and stability) and sialyl-Lewis X/a epitope (a major ligand for E-selectin) were more abundant at Asn 41. These results indicate that N-glycans attached to Asn 6 (protecting HAPLN1) and Asn 41 (supporting molecular interactions) play different roles in HAPLN1. This is the first study of site-specific N-glycosylation in rhHAPLN1, which will be useful for understanding its molecular interactions in the extracellular matrix.