N-glycosylation in Spodoptera frugiperda (Lepidoptera: Noctuidae) midgut membrane-bound glycoproteins

Abstract

Spodoptera frugiperda is a widely distributed agricultural pest. It has previously been established that glycoproteins in the midgut microvillar membrane of insects are targets for toxins produced by different organisms as well as plant lectins. However, there is still little information about the N-glycome of membrane-bound midgut glycoproteins in Lepidoptera and other insect groups. The present study used mass spectrometry-based approaches to characterize the N-glycoproteins present in the midgut cell microvilli of Spodoptera frugiperda. We subjected midgut cell microvilli proteins to proteolytic digestion and enriched the resulting glycopeptides prior to analysis. We also performed endoglycosidase release of N-glycans in the presence of H₂¹⁸O determining the compositions of released N-glycans by MALDI-TOF MS analysis and established the occupancy of the potential N-glycosylation sites. We report here a total of 160 glycopeptides, representing 25 N-glycan compositions associated with 70 sites on 35 glycoproteins. Glycan compositions consistent with oligomannose, paucimannose and complex/hybrid N-glycans represent 35, 30 and 35% of the observed glycans, respectively. The two most common N-glycan compositions were the complex/hybrid Hex₃HexNAc₄dHex₄ and the paucimannose structure that contains only the doubly-fucosylated trimannosylchitobiose core Hex₃HexNAc₂dHex₂, each appearing in 22 occupied sites (13.8%). These findings enlighten aspects of the glycobiology of lepidopteran midgut microvilli.

Keywords: Aminopeptidase N; Glycoproteins; Glycoproteomics; Midgut microvillar membrane; Spodoptera frugiperda; Transferrin.

Figure 1:

Schematic workflow for glycomic and glycoproteomic experiments using midgut microvilli preparations. *Glycosidase A treatment performed in presence or absence of H₂¹⁸O.

Figure 2:

SDS-PAGE of midgut microvilli. Except in region 2, the numbered regions were cut in >2-mm bands and individually digested, with subsequent pooling of fractions for analyses. The dashed lines in R indicate the 2-mm bands (named A to F) that were individually analyzed. BBMV, brush border membrane vesicles. R: schematic representation of how the gel was cut and pooled for analyses. S: molecular mass standards (kDa).

Figure 3:

Manual interpretation of the MS2 spectrum of a glycopeptide scanned in the Q Exactive HF. Upper plot) Spectrum overview. Lower plot) Expansion of the range m/z 17–1300 to highlight peaks used for de novo sequencing of glycopeptide LIQNQPINATR⁴⁶⁶. Hex: hexose. HexNAc: N-acetylhexosamine.

Figure 4:

Glycopeptide sequencing by PEAKS plus manual interpretation of the spectra. The MS2 spectra were obtained with the Q Exactive HF (A) and with the 6550 Q-TOF (B). The labels in green refer to the masses of the oxonium ion, peptide, peptide + HexNAc and peptide + HexNAc₂ and were added manually. HexNAc: N-acetylhexosamine. The data sheets for all glycopeptides identified in this work are available in Supplementary file 1.

Figure 5:

Sequence pattern analysis of occupied asparagines present in the sequons of identified glycopeptides. The percentage of difference is related to a random peptide database generated based on Bombyx mori genome. Amino acid position reflects the alignment anchored in the occupied asparagine for sequence pattern of different glycopeptides, and not its real position in the protein.

Figure 6:

MALDI-TOF analysis of permethylated N-glycans released after glycosidase A treatment in the presence of H₂¹⁸O. Prior to this, microvillar membrane proteins were in-solution digested.