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

https://doi.org/10.1016/j.cbpb.2020.110464Get rights and content

Highlights

  • A sum of 160 glycopeptides were sequenced, representing 25 N-glycan compositions associated to 70 sites on 35 glycoproteins.

  • Glycan compositions are represented by oligomannose (35%), paucimannose (30%) and complex/hybrid N-glycans (35%).

  • The two most common N-glycan compositions were Hex3HexNAc4dHex4 and paucimannose Hex3HexNAc2dHex2, each appearing in 22 occupied sites.

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.

Introduction

The cell surfaces of different tissues and organisms display glycoconjugates that consist of carbohydrates covalently linked to proteins and lipids. In mammals, these glycoconjugates are involved in a variety of functions, including host-pathogen interactions, aspects of the immune response, control of enzymatic activity, cell signaling and cell-cell attachment (Dwek, 1996; Varki, 2017). Most of these roles are also observed in insects, as reviewed by Walski and collaborators (Walski et al., 2017b). In cell-surface glycoproteins, modifications of asparagine residues by N-glycosylation and of serine, threonine or tyrosine residues by O-glycosylation are common. The complexity of carbohydrate assembly generates a diverse set of glycan modifications, in which the need of both instrument sensitivity and mass accuracy have been the major barriers to the use of analytical techniques like mass spectrometry (MS) for the investigation of glycan composition. However, in the last years, glycan analysis of glycoconjugates derived from complex samples has become more feasible with the development of sensitive mass spectrometric techniques (Han and Costello, 2013; Thaysen-Andersen and Packer, 2014).

The surfaces of lepidopteran midgut columnar cells are heavily glycosylated (Walski et al., 2017a); this is similar to the surface of human enterocytes, where, N-glycosylation plays an important role in the recognition and delivery of apical glycoproteins through a galectin-4 mediated mechanism in lipid rafts (Morelle et al., 2009). We previously described the presence of detergent-resistant membranes in the microvillar membrane of Spodoptera frugiperda midgut cells. This discovery suggested that lipid rafts may exist in lepidopterans, although the sorting mechanism had not yet been elucidated (Fuzita et al., 2019). Midgut cells are polarized, with both a basolateral membrane anchored to the gut epithelial basement membrane and an apical region with many microvilli that have a large surface area to maximize contact with the contents of the gut. In insects, as in other organisms, microvilli are involved in physiological processes, including digestion, nutrient and water absorption and water secretion (Terra and Ferreira, 2012).

The basolateral membrane is involved in cell-cell interactions. In contrast, midgut microvilli act as selective cellular gatekeepers. Thus, microvillar proteins deal with homeostasis control, food digestion and nutrient absorption and are frequently the targets of toxins and pathogens that make their way to the midgut lumen. The insecticidal activities of Cry endotoxins produced by the entomopathogenic bacterium Bacillus thuringiensis and some plant lectins have been attributed to the binding of these agents to midgut membrane proteins (Bravo et al., 2004; Budatha et al., 2007; Lagarda-Diaz et al., 2016; Zhang et al., 2006). Glycans displayed on membrane glycoproteins may interact with Cry toxins and other uncharacterized toxins. As an example, it is known that domain III from Cry1Ac specifically recognizes N-acetylgalactosamine in different membrane-bound proteins and enhances pore formation by the pre-pore toxin oligomer (Kitami et al., 2011; Masson et al., 1995; Pardo-Lopez et al., 2006). Other examples of similar interactions have been observed for aminopeptidase N (APN) (Bravo et al., 2004; Luo et al., 1997), alkaline phosphatase (ALP) (Jurat-Fuentes and Adang, 2004; Ning et al., 2010; Perera et al., 2009; Sarkar et al., 2009) and an uncharacterized 270-kDa glycoconjugate (Valaitis et al., 2001). Plant lectins can also bind to midgut membrane proteins and exhibit insecticidal activity, as observed for ferritin (Du et al., 2000), aminopeptidase (Cristofoletti et al., 2006) and amylase (Lagarda-Diaz et al., 2016).

Currently, there is limited information available on midgut microvillar glycoproteins. Most of research reported to date was not specific to the midgut (Stanton et al., 2017; Vandenborre et al., 2011). In the case where midgut cells were used, the glycans were evaluated by lectin binding (Walski et al., 2017a). Although an important knowledge about cell polarization and glycan distribution was generated by this latter research, this type of study does not allow to discover the exact full glycan compositions, determine the occupancy of potential glycosylation sites on the proteins or to discover their sequences. Therefore, characterization of the midgut microvillar glycoproteins by other techniques is crucial for complementing information on this underexploited field. Our goal in the research reported herein was use mass spectrometry analyses to determine the site-specific compositions of N-glycans released from Spodoptera frugiperda midgut microvillar glycoproteins. We determined the compositions of a total of 25 different N-glycans that are associated with 70 occupied N-glycosylation sites that we identified on 35 glycoproteins; these summed to a total of 160 unique glycopeptides.

Section snippets

Animal care and collection of midgut microvilli

S. frugiperda were reared as previously described (Parra, 1986). Larvae were individually housed in glass vials with a diet of beans from Phaseolus vulgaris, wheat germ, yeast and agar. S. frugiperda were maintained with a natural dark/light regime at 25 °C. During their last instar, 25 larvae were sacrificed and the midgut microvilli were obtained as previously described (Capella et al., 1997).

Proteomic, glycomic and glycoproteomic analyses

The analytical workflow used for preparation and analysis of S. frugiperda peptides, glycopeptides

Proteins identified in S. frugiperda midgut microvilli

The workflow used to investigate the N-glycans present in midgut microvilli is shown (Fig. 1). SDS-PAGE separation of midgut microvilli extracts was performed prior to protein digestion to 1) decrease the sample complexity, 2) obtain approximate molecular weight information, 3) improve tryptic digestion of membrane proteins and 4) minimize the impact of proteases and protease inhibitors present in midgut microvilli. The proteins were analyzed after in-solution or in-gel digestions via

Conclusions

In this study, we assigned 160 N-glycopeptides, representing 25 N-glycan compositions associated with 70 sites on 35 glycoproteins derived from S. frugiperda midgut microvilli preparations. Many of the glycoproteins have multiple occupied N-glycosylation sites and display more than one glycoform (microheterogeneity). Complex/hybrid glycans constituted 35% of the assigned glycopeptides, a finding that differs from what has been observed in other studies of glycosylation in insects. This is the

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Institutes of Health (grant numbers P41 GM 104603, S10 OD021651 and S10 OD010724) and the São Paulo Research Foundation- FAPESP (grant numbers 2014/14183-2, 2016/09511-6 and 2011/51685-8).

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