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Glycoproteins in the Midgut Microvilar Membrane of Spodoptera frugiperda (Lepidoptera: Noctuidae)
Author(s) -
Fuzita Felipe J,
Chandler Kevin B,
Haserick John R,
Ferreira Clélia,
Terra Walter R,
Costello Catherine E
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.784.6
Subject(s) - midgut , spodoptera , glycoprotein , glycan , chemistry , cry1ac , biochemistry , lysis , lepidoptera genitalia , bacillus thuringiensis , microbiology and biotechnology , chromatography , biology , bacteria , botany , larva , gene , recombinant dna , genetically modified crops , transgene , genetics
Spodoptera frugiperda , the fall armyworm, is an agricultural pest widely distributed from South to North America. The use of specific and environmentally friendly pesticides such as Cry toxins produced by the bacteria Bacillus thuringiensis is an important way to control these animals. There is evidence that these toxins bind to membrane‐bound proteins (named Cry toxin receptors‐CTR) of the midgut microvilar epithelium (MME), leading to cell lysis or activating cell death pathways. Among the recognition mechanisms, it has been reported that domain III of Cry1ac interacts with glycans attached to CTR, and that the presence of N ‐acetylgalactosamine enhances pore‐formation activity. Nevertheless, only one published report describes glycan structures present for the CTR aminopeptidase N (APN). Moreover, there is still a huge lack of information about glycan composition in insects, which may differ in composition from mammalian ones. Thus, this study aims to do a broad characterization of glycoproteins membrane‐bounded to the midgut microvilar epithelium of S. frugiperda . Isolated MME was submitted to both in‐gel and in‐solution tryptic digestions. Gel band digests were separately submitted either to online HILIC/C18 LC‐MS/MS in an Agilent 6550 Q‐TOF/TOF instrument or to reversed phase LC‐MS/MS into a Q Exactive HF (Thermo). A fraction of each of the same gel band digests was treated with Glycosidase A and analyzed in the latter instrument. Permethylation of released glycans was done after in‐solution digestion and treatment with Glycosidase A in the presence of H 2 18 O. Further analysis of permethylated glycans was accomplished by MALDI‐MS in an UltrafleXtreme (Bruker). Spectra assignments were done either manually or using Peaks ® 8 (BSI). A total of 16 different glycoproteins (21 glycopeptides) were identified in S. frugiperda MME, including two unknown proteins. Four APNs and one alkaline phosphatase, already known CTRs, are among the identified glycoproteins. In more than one case, an APN glycopeptide showed multiple glycoforms. For instance, peptide 99 INVV N NTNGENVQLR 113 was identified with Hex 9 HexNAc 2 , Hex 3 HexNAc 2 dHex 5‐6 , Hex 4 HexNAc 3 dHex 2 , among others. Transferrin was shown to be heavily glycosylated, displaying four different glycopeptides. The most commonly observed structure was the high mannose glycan (peptides 456 LIQNQPI N ATR 466 and 624 ALCR N NSLALR 634 ), but fucosylated glycans Hex 3 HexNAc 2 dHex 2 and Hex 4 HexNAc 2 dHex 2 were found in peptides 473 ESSVVSG N VSR 483 and 241 VNA N ATNTAVAYVAWQHVR 259 , respectively. The assignments of most abundant glycan structures were confirmed after release and permethylation followed by MALDI‐MS analysis. Novel aspect: This is the first broad characterization of glycoproteins present in the midgut of a lepidopteran which will allow a better understand of possible Cry toxin targets and improve the knowledge about invertebrate glycans. Support or Funding Information Supported by FAPESP (2014/14183‐2; 2016/09511‐6; 11/51685‐8) and NIH (P41 6M104603)