Site-specific N-glycosylation differences between two recombinant human hyaluronan and proteoglycan link protein 1 forms with distinct hyaluronan-proteoglycan linking activity.
The study found that rhH-1 has a 7.5-fold higher linking potency toward hyaluronan and proteoglycans compared to rhH-2, attributed to distinct N-glycosylation differences.
Where it sits
this study against the rest of the mgf (mechano growth factor) corpusSummary and findings
This study characterized two recombinant human hyaluronan and proteoglycan link protein 1 (HAPLN1) forms expressed in CHO cells. The linking potency of rhH-1 was found to be 7.5-fold higher than that of rhH-2, with EC50 values of 133.5 ng/mL and 994.8 ng/mL, respectively. The study highlights distinct site-specific N-glycosylation differences between the two forms.
Abstract
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 (Asn<sup>6</sup> and Asn<sup>41</sup>), 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 Asn<sup>6</sup>, 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, Asn<sup>41</sup> 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 (EC<sub>50</sub> 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<sub>max</sub>). 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.