Structural determination of an N-glycan moiety attached to the prothoracicotropic hormone from the silkmoth Bombyx mori

Figures & data

Fig. 1. Analyses of peptides derived from the native and recombinant PTTHs.

Notes: (A) Comparison of the RP-HPLC profiles of lysyl endopeptidase-treated E-rPTTH and B-rPTTH. Peaks A in E-rPTTH and B in B-rPTTH were eluted at different retention times. The peptides corresponding to peaks A and B were further analyzed to determine the structure of the N-glycan. (B) MALDI-TOF MS analyses of the peptides derived from B-rPTTH and E-rPTTH. A possibly N-glycosylated peptide derived from B-rPTTH (a) and E-rPTTH (b) was analyzed. After PNGase F digestion, a peptide corresponding to peak B in Fig. 1(A) was also analyzed (c). The asterisk indicates an ion peak corresponding to the molecular weight of an N-glycosylated peptide without a deoxyhexose, possibly a fucose residue.

Fig. 2. Structural analyses of PA-N-glycan on PTTHs.

Notes: (A) 2D PA-N-glycan map. The dots indicate the PA-N-glycan derived from B-rPTTH. The arrows indicate the migration on the map after treatment of the PA-N-glycan with α-fucosidase and α-mannosidase. Mapped glucose units were defined by the retention times of the commercially available PA-glucose oligomers. (B) HPLC profiles of PA-N-glycans derived from native PTTH and B-rPTTH using ODS column. Peaks corresponding to PA-N-glycans indicate numbers on peaks. (C) Structures of N-glycan on PTTHs. The percentages of N-glycans are indicated by each fluorescent peak intensity of PA-derivatives observed in (B).

Fig. 3. Biological activities of the native and recombinant PTTHs.

Notes: Dose-dependent activity was observed using three different PTTHs. The biological activity of PTTH was evaluated using the average of the apolysis index (N = 7); points 4, 3, 2, or 1 were observed if apolysis was observed on the 3rd, 4th, 5^th, or 6th days after sample injection into the brainless pupae, respectively.⁶⁾ Closed circles, open circles, and open triangles indicate the effects of native PTTH, B-rPTTH, and E-rPTTH, respectively. The biological activities of these PTTHs were shown to be reproducible using a different preparation of brainless pupae.

Kataoka H, Nagasawa H, Isogai A, Tamura S, Mizoghuchi A, Fujiwara Y, Suzuki C, Ishizaki H, Suzuki A. Agric. Biol. Chem. 1987;51:1067–1076.10.1271/bbb1961.51.1067

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