The presence of monoglucosylated N196-glycan is important for the structural stability of storage protein, arylphorin

Although N-glycosylation has been known to increase the stability of glycoproteins, it is difficult to assess the structural importance of glycans in the stabilization of glycoproteins. APA (Antheraea pernyi arylphorin) is an insect hexamerin that has two N-glycosylations at Asn¹⁹⁶ and Asn³⁴⁴ respectively. The glycosylation of Asn³⁴⁴ is critical for the folding process; however, glycosylation of Asn¹⁹⁶ is not. Interestingly, the N196-glycan (glycosylation of Asn¹⁹⁶) remains in an immature form (Glc₁Man₉GlcNAc₂). The mutation of Asn¹⁹⁶ to glutamine does not change the ecdysone-binding activity relative to that of the wild-type. In the present study, we determined the crystal structure of APA, and all sugar moieties of the N196-glycan were clearly observed in the electron-density map. Although the sugar moieties of the glycan generally have high structural flexibility, most sugar moieties of the N196-glycan were well organized in the deep cleft of the subunit interface and mediated many inter- and intrasubunit hydrogen bonds. Analytical ultracentrifugation and GdmCl (guanidinium chloride) unfolding experiments revealed that the presence of the N196-glycan was important for stabilizing the hexameric state and overall stability of APA respectively. Our results could provide a structural basis for studying not only other glycoproteins that carry an immature N-glycan, but also the structural role of N-glycans that are located in the deep cleft of a protein.