Site-specific N-glycosylation differences between two recombinant human hyaluronan and proteoglycan link protein 1...

Hyaluronan and proteoglycan link protein 1 (HAPLN1) is a glycoprotein that stabilizes hyaluronan-proteoglycan complexes and maintains extracellular matrix integrity. Two protein forms of HAPLN1 have been reported; however, their structural and functional distinctions remain poorly understood. Here, we characterized two recombinant human HAPLN1 (rhHAPLN1) forms expressed in CHO cells using in-gel glycomic and glycoproteomic analyses with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nano-LC-MS/MS. SDS-PAGE resolved two protein forms: rhH-1 (∼44 kDa, 76.3%) and rhH-2 (∼40 kDa, 23.7%). Both forms exhibited identical amino acid sequences and shared two N-glycosylation sites (Asn⁶ and Asn⁴¹), as confirmed by analysis of in-gel tryptic glycosylated and deglycosylated peptides. A total of 22 and 15 N-glycans were identified in rhH-1 and rhH-2, respectively. At Asn⁶, rhH-1 contained highly branched, sialylated N-glycans (50.0%, normalized to 100% for each site), whereas rhH-2 showed low occupancy (3.7%) with non-sialylated N-glycans. In contrast, Asn⁴¹ exhibited nearly identical profiles in both forms, comprising predominantly bi-antennary, highly sialylated N-glycans (84.0% in rhH-1 and 83.9% in rhH-2). Mild formic acid preserved tri/tetra-sialylation during N-glycopeptide recovery, and higher-energy collisional dissociation at two normalized collision energies improved N-glycan/peptide fragmentation for confident N-glycopeptide identification. Functional assays demonstrated that rhH-1 displayed 7.5-fold higher linking potency (EC₅₀ values of 133.5 ng/mL for rhH-1 and 994.8 ng/mL for rhH-2) toward hyaluronan and proteoglycans than rhH-2, while both showed similar efficacy (E_max). This study provides the first comprehensive structural and functional comparison of sequence-identical rhHAPLN1 forms, revealing distinct site-specific N-glycosylation associated with differences in potency.