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.
Magnetic Resonance Team, Korea Basic Science Institute, Gwahangno 113, Daejeon 305-333, South Korea.
Kyoung-Seok
KS
Ryu
Jie-Oh
JO
Lee
Taek Hun
TH
Kwon
Han-Ho
HH
Choi
Hong-Seog
HS
Park
Soo Kyung
SK
Hwang
Zee-Won
ZW
Lee
Kyung-Bok
KB
Lee
Young Hyun
YH
Han
Yun-Seok
YS
Choi
Young Ho
YH
Jeon
Chaejoon
C
Cheong
Soohyun
S
Kim
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D004440:Ecdysone / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding
D019476:Insect Proteins; D011134:Polysaccharides; C041137:storage proteins, Insecta; D004440:Ecdysone