Total chemical synthesis of homogeneous folded, misfolded, and productive/nonproductive intermediate form of N-glycosylated crambin for investigation of UGGT recognition
In order to better understand whether UGGT also recognize and facilitate the refolding of folding intermediate, based on previous research, the Kajihara group synthesized the N-glycosylated protein crambin bearing M9-high mannose type N-glycan 104 for investigation of UGGT’s recognition.[33] Glycosyl crambin was a suitable substrate for this research because the folding process only yielded correctly folded glycoprotein as a sole product. The sequence of crambin was divided into two segments: segment 101 (sequence:1-15) and segment 102 (sequence:16-46, glycosylation: N17). After SPPS to obtain both segments, the NCL afforded a full length glycosylated crambin 103 bearing M9 N-glycan which is folded to give native glycosyl crambin 104. To prepare misfolded crambin 105 and unproductive intermediate, the native Cys3 or Cys4 were mutated into Ser to hamper the disulfide formation. Air oxidation folding in the absence of redox reagents afforded three kinds of misfolded glycosylated crambin 105 through formation of unproductive intermediates. (Fig.12A, top) With correctly native sequence and mutated sequence M9-glycosyl crambin in hand, the UGGT binding assay was tested using these substrates which yield G1M9-glycoprotein as product 109. The result was analyzed by LC/MS according to the MS intensity of G1M9-glycoprotein. (Fig.12B, bottom) According to the result, both productive folding intermediate during correct folding of native sequence, and unproductive intermediate during misfolding of mutated sequence were recognized by UGGT to afford G1M9 product. In this research, the authors were able to elucidate the recognition pattern of UGGT toward folding intermediates.
