Glycoproteins in the Midgut Microvilar Membrane of Spodoptera frugiperda (Lepidoptera: Noctuidae)
Authors: Fuzita Felipe J , Chandler Kevin B , Haserick John R , Ferreira Clélia , Terra Walter R , Costello Catherine E 
Published: November 2023

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 H218O. 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 99INVVNNTNGENVQLR113 was identified with Hex9HexNAc2, Hex3HexNAc2dHex5‐6, Hex4HexNAc3dHex2, among others. Transferrin was shown to be heavily glycosylated, displaying four different glycopeptides. The most commonly observed structure was the high mannose glycan (peptides 456LIQNQPINATR466 and 624ALCRNNSLALR634), but fucosylated glycans Hex3HexNAc2dHex2 and Hex4HexNAc2dHex2 were found in peptides 473ESSVVSGNVSR483 and 241VNANATNTAVAYVAWQHVR259, 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 InformationSupported by FAPESP (2014/14183‐2; 2016/09511‐6; 11/51685‐8) and NIH (P41 6M104603)