Precision mapping of N- and O-glycoproteins in viral resistant and susceptible strains of Bombyx mori

Journal of Invertebrate Pathology 167 (2019) 107250

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Precision mapping of N- and O-glycoproteins in viral resistant and susceptible strains of Bombyx mori

T

Feifei Zhua,b, Dong Lia, Dandan Songa, Hengchuan Xiaa, Xiaoyong Liua, Qin Yaoa, ⁎ Keping Chena,b, a b

Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Silkworm Bombyx mori Glycosylation mapping Glycan Viral resistance

Protein glycosylation plays important roles in protein structure, function, and immune recognition, among many other activities. One of the major roles of glycans and glycoconjugates on the cell surface is acting as the receptor for outside pathogens such as viruses. During the initial stage of viral replication, viruses interact with cell membrane receptors, which are in many cases glycoproteins. Identifying such glycoproteins is essential to understanding the mechanisms of viral infection, as well as developing antiviral strategies. Silkworm is an important economic insect as well as a model organism for molecular biology, yet current knowledge on its glycoproteome is far from complete due to both analytical challenges and perceived lack of importance. In this study, we performed a large-scale glycoproteomic survey for two silkworm Bombyx mori strains 306 and NB, which are susceptible and resistant to the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), respectively. More than 400 silkworm N- and O- glycoproteins were identified with high confidence, demonstrating that this organism employs extensive glycosylation. We mapped some glycoproteins only to the BmNPV susceptible or resistant strain, underlining the potential relationship between glycosylation and viral susceptibility. We predicted O-glycoproteins and O-glycan compositions for the first time for this organism. The variations in glycan site occupancy, as well as glycan diversity between the two silkworm strains, provide an insight into role of glycosylation in viral recognition and infection processes.

1. Introduction Glycosylation is one of the most common and perhaps the most diverse post-translational modification to eukaryotic proteins. This modification has been associated with many critical biological processes, although in many cases the functions of specific protein glycosylation are yet to be determined (Varki, 2016). Glycans and glycoconjugates decorating the cell surface can react to outside pathogens such as viruses. At the initial stage of viral infection, the virus interacts with cell surface receptors, which are in many cases membrane glycoproteins (Sugrue, 2007). Therefore, a particular type of glycan that exists in a cell membrane can determine host susceptibility to a viral pathogen (Bagdonaite and Wandall, 2018). There also has been evidence that certain viral infections may alter host glycosylation profiles by overexpression of specific glycoenzymes. For example, it previously was shown that baculoviruses infected Lepidoptera larvae express a Glc-transferase, adding Glc to the molting hormone ecdysone, which inactivates the hormone and thereby prevents the metamorphosis of the

⁎

caterpillar host (Reilly and Miller, 1989). Compared to mammalian glycobiology which is undergoing extensive study, insect glycobiology is frequently being neglected, partially due to the lack of anticipated importance. In recent years, however, investigations of the glycobiolgy of model insects such as Drosophila have demonstrated the essential and broad range of activities of various glycoconjugates in invertebrate organisms (Walski et al., 2017; Zhu et al., 2019). Emerging as a new model insect, the silkworm Bombyx mori is not only a valuable economic insect in many developing countries, but also an established model organism for fundamental biological research (Meng et al., 2017). Currently, knowledge about the silkworm glycoproteome is very limited. To date, only a handful of glycoproteins have been studied and glycan modifications reported, and even less is known about the functions of the silkworm glycoproteins. Currently, only a limited number of insect species has been mapped for their complete glycoproteomes (Zielinska et al., 2012). About 50 glycoproteins have been reported in B. mori to date, including arylphorin, actin, sericin and hemocyanin (Sinohara, 1979; Kim et al., 2003; Hiro

Corresponding author. E-mail address: [email protected] (K. Chen).

https://doi.org/10.1016/j.jip.2019.107250 Received 9 July 2019; Received in revised form 16 September 2019; Accepted 17 September 2019 Available online 18 September 2019 0022-2011/ © 2019 Published by Elsevier Inc.

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analysis system.

et al., 2006; Vandenborre et al., 2011). However, the sites of glycosylation on these proteins and the specific glycan structures remain ambiguous or unidentified, and the roles of glycosylation on these proteins have not been clarified. Although the importance of protein glycosylation has been widely recognized, obtaining the glycoproteome information is still a challenging task due to the low levels and extreme diversity of glycosylated proteins in an organism. A mass spectrometry-based, precision mapping of N-glycosylation sites in an organism was developed by Zielinska et al. (2010) by database searching against asparagine mass modification of 2.988 Da due to deamidation in 18O water. Using this method, they mapped N-glycoproteomes across different species, including insects, and found that N-glycoproteins are significantly overrepresented in the cell membrane and play pivotal roles in cell-cell communication, organ development, and body growth (Zielinska et al., 2012). We employed a similar strategy for large scale N-glycosylation mapping of the model organism silkworm Bombyx mori and, due to reduced complexity upon removal of N-glycans by deamination of enriched (glyco)peptides, we predicted O-glycosylation sites and compositions by setting variable modifications to serine or threonine residues against a panel of common insect O-glycans. To gain an insight into the relationship between glycosylation and potential antiviral activity, we compared the glycoproteomes between B. mori strains 306 and NB, which are susceptible and resistant to the Bombyx mori nucleopolyhedrovirus (BmNPV), respectively. Strain NB has 1000-fold LD50 than Strain 306 for the virus [unpublished observation]. The differences at the proteomic level between the two strains was reported previously (Liu et al., 2010; Qin et al., 2012), however, increasing awareness that protein glycosylation is fundamental to a protein’s structure and function makes it necessary to look at the detailed glycosylation map in addition to the regular proteomic level. This study represents the first comprehensive mapping of glycoproteome in the insect B. mori, and underlines the potential relationship between glycosylation and insect antiviral activity.

2.3. Filter-aided tryptic digestion, glycopeptide enrichment, and deamination

18

O

The filter aided 18O deamination was performed according to the published protocol with modifications (Zielinska et al., 2010). An aliquot of protein extract containing 400 µg total proteins were measured out and boiled for 5 min, and then cooled to room temperature before adding 200 µL UA buffer (8 M urea, 150 mM Tris-HCl, pH 8.0). Samples were mixed and centrifuged in a 10-kDa ultrafiltration centrifuge tube (Millipore) at 14,000g at 4 °C for 15 min. The filtrate was discarded and this step was repeated before 100 µL of 100 mM iodoacetamide in UA buffer was added. Samples were mixed at 600 rpm for 1 min and incubated for 20 min in darkness. After 15 min centrifugation at 14,000g at 4 °C, the filters were washed three times with UA buffer. Then 100 µL 25 mM NH4HCO3 were added to the filters and the samples were centrifuged for 15 min at the same conditions as before. This step was repeated twice. Samples were added with 40 µL trypsin buffer (trypsin: protein = 1:50) and gently mixed for 1 min, and then incubated at 37 °C for 16 h. Samples were placed in new tube to collect the filtrate. The filter was washed with 40 µL 25 mM NH4HCO3 and the filtrate was combined. The digested peptides were transferred to a 10-kDa filter and 100 μL lectin mixture containing 2.5 mg/mL Con A, 2.5 mg/mL WGA, and 0.8 mg/mL RCA in buffer A (buffer A = 1 mM CaCl2, 1 mM MnCl2, 0.5 M NaCl in 20 mM Tris-HCl, pH 7.3) was added and mixed at 600 rpm for 1 min before incubation at room temperature for 1 h. The samples were centrifuged at 14,000g for 10 min and washed twice by 200 µL buffer A. Then 50 µL 25 mM NH4HCO3 in H218O was added and discarded by centrifugation. This step was repeated twice. The filter was placed in a new collection tube and 40 µL PNGase F (500 units) in 25 mM NH4HCO3 made in H218O was added to the filter and the samples were incubated for 3 h at 37 °C. The deglycosylated peptides were eluted by centrifugation and the filter was washed with 25 mM NH4HCO3 in H218O twice and the elutions combined. The samples were then desalted using a zip-tip protocol according to the manufacture’s protocol and ready for LC-MS analysis.

2. Experimental 2.1. Silkworm breeding and protein extraction Silkworm strains NB and 306 were reared on fresh mulberry leaves at 25 °C and 70–90% relative humidity. On the first day of fifth instar, silkworms were harvested, washed with phosphate buffered saline (PBS) and residual mulberry leaves removed from the gut. To the silkworm sample, 1.5 mL SDT lysis buffer (4% SDS (w/v), 100 mM Tris/ HCl, 0.1 M DTT, pH 7.6) was added, and the sample was homogenized in a tissue homogenizer (Shanghai Jingxin Co., Shanghai, China) using three beating cycles at 120 Hz, 60 s at 4 °C. The solution was sonicated in an ice bath for 10 min and then boiled for 15 min before centrifuging for 40 min at 14,000 g. Supernatants were collected and protein concentrations determined by the Bradford assay (ThermoFisher Scientific), and stored at −80 °C for further use.

2.4. Mass spectrometry analysis The deaminated peptides were loaded onto a Acclaim PepMap100 column (nanoViper C18, 100 μm * 2 cm, Thermo Scientific) and then further separated by a C18 capillary column (75 μm * 10 cm, 3 µm). Buffer A was 0.1% formic acid and buffer B was 0.1% formic acid in 84% ACN. Mixed peptides were separated at a flow rate of 250 nL/min using following gradient: (solvent B, 0–2 min from 4% to 10%; 2–122 min from 10% to 20%; 122–240 min from 20% to 45%; 240–250 min from 45% to 100%; 250–270 min at 100%). The separated peptides were analyzed on a LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher). After each full scan, the top 10 intense precursor ions were isolated and fragmented using HCD at an isolation window of 2 m/ z and normalized collision energy of 30 eV.

2.2. SDS-PAGE and lectin blot The extracted silkworm proteins were analyzed on a 12% SDS-PAGE gel, and stained with Coomassie brilliant blue for 2 h and subsequently de-stained for 12 h. The gel was then electrotransferred onto a PVDF membrane (0.45 µm) at 220 mA for 60 min at 4 °C. The membrane was blocked with Carbo-Free™ Blocking Solution (Vector Laboratories) in sufficient volume for 30 min at room temperature, then incubated in PBS containing approximately 2–20 µg/ml biotinylated lectin for 30 min and washed with TPBS (PBS + 0.05% Tween 20) for 5 min. The streptavidin-conjugated horseradish peroxidase was added and incubated for 30 min before washed with TPBS for 5 min. Peroxidase substrate High-sig ECL (Tanon Sci & Tech Co., Shanghai, China) was used to detect chemiluminescent signal using Tanon 5200 Multi

2.5. Data analysis Protein identification and N-glycosylation site mapping were analyzed by Peaks Studio software (Bioinformatics Solutions Inc.). Silkworm reference proteome was downloaded from Uniprot (Proteome ID UP000005204). Two missed cleavages were allowed by tryptic digestion, carbamidomethyl was set as fixed modification, and methionine oxidation and protein N-terminal acetylation were set as variable modifications. N-glycosylation was mapped by setting variable modification of +2.988 Da on the asparagine residue. O-glycosylation was analyzed by searching against a panel of 37 O-glycan modifications on the threonine or serine residues (Table S7). The maximum number of 2

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3.2. A significant portion of silkworm N-glycoproteins are transmembrane proteins

modifications per peptides was set as 7 and main search mass tolerance was 5 ppm. A peptide false discovery rate of 0.01, de novo ALC score of 50%, PTM Ascore of 20, and protein unique peptides of 2 were used to filter the mapping results. Uniquely mapped glycoprotein to a particular strain was obtained by comparing the total identified glycoproteins between the two strains (Tables S1 and S2). Protein topology analysis was analyzed using Phobius web server (Käll et al., 2007). Protein with a transmembrane helices probability score greater than 80% was considered to be a transmembrane protein. Protein subcellular localization was predicted using CELLO2GO web server (Yu et al., 2014). Gene Ontology enrichment analyses for overrepresented cellular components were performed using ShinyGO webserver (Ge and Jung, 2018).

Cell membrane proteins are frequently decorated with glycans, which can initiate a first surface response to pathogens (Kupferschmied et al., 2016). Barrows et al. (2007) reported that C. elegans cell surface glycans influence bacteria adhesion to the host, which is usually the first step during bacterial infection. Therefore, it is important to examine the membrane glycoprotein populations to discover potential cell surface glycoconjugate receptors during pathogen invasions. Predicted subcellular localization analysis showed that a quarter of the identified N-glycoproteins are located at the plasma membrane, whereas other major localizations include extracellular region, cytoplasmic and nuclear compartment (Fig. 2A). An important criterion for the classification of transmembrane proteins is the signal peptide, which basically determines the transmembrane topology of the protein (Kaji et al., 2007). To this end, the membrane glycoproteins are further classified based on the number of transmembrane domains and the presence of signal peptide, which can locate the protein into the lumen of the endoplasmic reticulum before the initiation of N-glycosylation. Fig. 2B shows that of the 113 transmembrane N-glycoproteins (full list shown in Table S4), a predominant portion has a single transmembrane domain, and more than half of the glycoprotein does not have a signal peptide. Kaji et al. (2007) previously reported that for signal peptide-containing, single domain transmembrane glycoproteins, the glycosylation sites are almost exclusively located on the N terminal region of the transmembrane protein, which were classified as type I transmembrane glycoprotein. For membrane proteins that do not have a signal peptide, a signal anchor sequence plays an equivalent role in directing the protein to the ER, only differing in that a signal peptide is cleaved off from the protein but the signal anchor remains intact (Kaji et al., 2007). In this case, the glycosylation sites can be either at the N-terminal or C-terminal region of the transmembrane protein.

3. Results and discussion 3.1. Global mapping reveals a large number of N-glycoproteins in silkworm To ensure maximum recovery of the (glyco) peptides, an LC gradient of over 4 h was used, and the mapping results were filtered using a highly stringent filter. A total of 409 glycoproteins were identified in the silkworm (Tables S1 and S2), demonstrating that the silkworm species contains a surprisingly large N-glycoproteome. Compared to Drosophila and Caenorhabditis, which have more than 2200 and 1400 Nglycosylation sites, respectively (Zielinska et al., 2012; Kaji et al., 2007), silkworm has fewer N-glycosylation sites, about 700 for each strain (Table S3). However, it is worth noting that when the N-glycosylation site is modified by α(1,3)-linked core fucose-containing glycans, this site may not be detected because such glycans are resistant to PNGase F digestion. Thus, the actual N-glycoproteome may be even larger. To assess the general glycosylation status of the silkworm proteins, lectin blot utilizing wheat germ agglutinin (WGA) and concanavalin A (Con A) lectins, which are specific to GlcNAc and mannose residues, respectively, were used to probe the general glycoprotein profile. GlcNAc and mannose are the N-glycan core components and also commonly are observed in O-glycans. The blot showed a dense population of glycoproteins in the range of 50 and 150 kDa (Fig. 1A). Glycoproteins detected by lectin blot appeared similar between the 306 and NB strains, but still had noticeable differences. We observed that GlcNAc containing glycoproteins are notably reduced in the 306 strain compared to the NB strain (Fig. 1A). This may be due to a decrease in the expression level of the glycoprotein or a to a reduced glycosylation site occupancy of the protein. The mannose-containing glycoprotein profiles appear essentially the same in the two stains. We noted that the lectin blot identifies and quantifies glycoproteins based solely on carbohydrate moiety. Thus, when a protein is multiply glycosylated or its glycan contains lengthy sugar residues, the protein appears to be more abundant. Likewise, when the protein is only singly glycosylated, and its sugar moiety is relatively simple, it may not be readily identified by the lectin method. Our data show that most low molecular weight glycoproteins (< 50 kDa) are singly or doubly glycosylated (Table S3), and were therefore observed as light smears in lectin blot (Fig. 1A). The lectin blot results were in agreement with the global glycosylation mapping statistics. Although the mapped glycoproteins spanned a wide molecular weight range from 10 to 800 kDa, most were distributed between 50 and 150 kDa (Fig. 1B). A total of 336 and 337 Nglycoproteins were mapped to the 306 and NB strains, respectively, with 264 glycoproteins shared by both strains (Fig. 1C). An average of two glycosylation sites per glycoprotein was estimated, and most glycoproteins tend to contain one, two, or three glycosylation sites; however, a few of the proteins can take more than five glycosylation sites (Fig. 1D).

3.3. Many N-glycoproteins are unknown or have not been characterized functionally or structurally Gene ontology enrichment in terms of biological processes shows that the mapped glycoproteins are mainly involved in metabolic processes and cell adhesion, which are significantly overrepresented in the N-glycoproteome compared to the total proteome (Fig. 3). This observation reinstates the important role of protein glycosylation in cell adhesion and metabolic pathways. However, a significant portion of the N-glycoproteins are uncharacterized proteins (Tables S1 and S2), indicating that our knowledge on silkworm glycoproteome are far from complete. In depth structural and functional characterization of specific proteins in silkworm, especially for those with glycosylation modifications, are necessary to fully understand the role of glycosylation and their functions. Among the most heavily N-glycosylated proteins, hemocytin is mapped with 9 N-glycosylation sites (Table S3). It is a major mediator of hemocyte aggregation, an important means of resisting bacterial infection (Arai et al., 2013). Another glycoprotein, carboxypeptidase, was mapped with 7 N-glycosylation sites (Table S3). Although this protein has not been functionally characterized in silkworm, its homologue protein was reported to contain 2 N-glycosylation sites in Helicoverpa armigera (Bown and Gatehouse, 2004). We noted that some of the identified glycoproteins, especially those with enzymatic activities such as α-mannosidase, ATPase, UDP-glucuronyltransferase, just to name a few, were also reported to be glycosylated in Drosophila and Caenorhabditis (Kaji et al., 2007; Zielinska et al., 2012), indicating a somewhat conserved glycosylation for certain proteins among different species. The storage protein arylphorin from another silkworm, Antheraea pernyi, previously was determined to be modified with 11 different N-glycans structures, however, the N-glycosylation sites were not elucidated (Kim et al., 2003). Our results show 3

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A

Con A lectin

kD

306

NB

306

306

NB

B

WGA A lectin

NB

160

# of proteins

170 130 100 70 55 40

35

140

NB

120

306

100 80 60 40

25

20 0

15

20 1 50 2100 3 150 4 200 5 250 6 300 7 350 8 4009 50010100011

A 306

72

D

120

264

NB

100

NB

# of proteins

C

Molecular weight (kD)

C

B

73

306

80 60 40 20 0 1

2

# of N-glycoproteins identified in each strain

3

4

5

>5

# of N-glycosylation site

Fig. 1. SDS-PAGE and lectin blot (A) of silkworm proteins; molecular weight distribution (B); number of mapped N-glycoproteins in each strain (C); and number of Nglycosylation sites (D) for the silkworm strains 306 and NB.

A

B

90

Nulcear 21%

Extracecullar 23%

Others 4%

# of N-glycoproteins

80

Mitochondrial 3%

No signal peptide

70

With signal peptide

60 50 40 30 20 10

cytoplasmic 24%

Plasmamembrane 25%

0

1

2

3

4

5

6

7

8

9

10

11

12

13

# of transmembrane domain Fig. 2. (A) Predicted subcellular localization of silkworm N-glycoproteins, (B) predicted transmembrane topology of silkworm N-glycoproteins.

was identified to be N-glycosylated at site N118. Other reported silkworm glycoproteins include BmApoD1 (Zhou et al., 2018), Bombyx mori prothoracicotropic hormone (Ishizaki and Suzuki, 1994), and 130-kDa glycoprotein (Shin et al., 2001); however, these were not identified in our study. Still, compared to the limited number of glycoproteins reported previously, our results indicate a far more complex glycoproteome that awaits further exploration.

that homologue of arylphorin in B. mori has 4 N-glycosylation sites, N72, N203, N211, and N214 in the strain 306 and N72 and N211 in the strain NB (Table S3). Trehalase, a 66-kDa sugar enzyme important for silkworm energy metabolism, was previously found to be glycosylated based on lectin blot assay (Ujita et al., 2011). We identified three versions of this glycoprotein (P32358, H9J822, H9J4Z6); with P32358 and H9J822 having similar N-glycosylation sites at N58 and N331, and H9J4Z6 at N48, N260, and N336 for both strains (Table S3). The silk glycoprotein sericin had two N-glycosylation sites (N51 and N226), in addition to one site that was previously reported (Sinohara, 1979). A recently characterized 27-kDa silkworm allergen (Jeong et al., 2016) 4

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Organic substance metabolic process Carbohydrate metabolic process Total proteome Biological adhesion

N-glycoproteome

Cell adhesion Protein metabolic process Metabolic process Organonitrogen compound metabolic process Proteolysis Carbohydrate derivative metabolic process 0%

10%

20%

30%

40%

Proportion of Proteins Fig. 3. Gene ontology enrichment analysis showing significantly overrepresented terms in biological functions for the silkworm N-glycoproteins compared to the total proteome.

3.4. Some N-glycoproteins are uniquely mapped to the BmNPV susceptible strain 306 or resistant strain NB

Table 1 N-glycoproteins uniquely mapped in strain 306 (partial).

Insect glycoconjugate profiles have been reported to be correlated with resistance to bacterial infections (Cipollo et al., 2004). Our results show that the BmNPV susceptible strain 306 and resistant silkworm NB appear to have different N-glycoproteome profiles. We found that over 20% of each glycoproteome for 306 and NB is unique, meaning that these glycoproteins are not present, or at least downregulated to a level not detectable in MS, in the opposite strain. Of the 72 N-glycoproteins unique to 306 (full list shown in Table S5), 27 have been identified at the protein, transcript, or gene level, or at least from homology (Table 1). Four of these unique N-glycoproteins are predicted to be transmembrane glycoproteins, which are of particular interest as they can be potential cell surface targets during viral infections. In fact, a 17 kDa glycoprotein with a predicted single transmembrane domain was recently cloned and characterized in the silkworm midgut epithelium and functions as cell-surface receptor for densovirus (Ito et al., 2018). Table 1 shows a partial list (those designated as characterized proteins in the Uniprot database) of uniquely mapped glycoproteins in strain 306. It is interesting to note that the glycoprotein dolichyl-diphosphooligosaccharide-protein glycosyltransferase, which is responsible for the initial transfer of the N-glycan precursor Glc3Man9GlcNAc2 from the lipid carrier to an asparagine residue (Das and Heath, 1980), and another glycoenzyme mannosyltransferase are transmembrane glycoproteins, each with two glycosylation sites. These two glycoenzymes were not mapped in the resistant strain, therefore, a change in the N-glycan profiles between the two species is expected. This observation also indicated the role of glycosylation for proper function of the glycoenzymes. For the BmNPV resistant strains, a total of 73 N-glycoproteins were mapped (full list in Table S6); however, none of 23 N-glycoproteins shown on the partial list in Table 2 are transmembrane proteins, indicating the possibility that cell surface glycoconjugates are lacking in the resistant strain. However, further

Protein ID

Mol. Weight (kDa)

Protein name

H9B457 Q5UAP4 H9JCR4 D8KY55 H9IUT1 H9JF44 Q2F6C2

28.5 33.4 33.4 67.4 78.0 54.3 57.6

H9ITR8 H9J305 B8XWD7 H9IZX9

43.7 105.9 10.5 45.2

Q1EPM0

35.4

H9JBJ6 F8UN44 O02387 H9J6Y5 O96052 P09334

147.5 72.7 15.3 15.4 11.0 29.7

H9JU51 H9J1A8 H9JFZ5 Q1HPY5 H9IT19

35.3 68.2 132.4 30.8 56.3

H9J072 H9JJJ8 H9JES9 Q1HPU3

18.1 58.8 25.3 16.9

30K protein 24 40S ribosomal protein SA 40S ribosomal protein SA Alpha amylase alpha-1 2-Mannosidase Carboxylic ester hydrolase Chaperonin containing t-complex polypeptide 1 beta subunit Chitinase-like protein EN03 Coatomer subunit beta' Death-related protein Dolichyl-diphosphooligosaccharide– protein glycosyltransferase 48 kDa subunit Glyceraldehyde-3-phosphate dehydrogenase Guanylate cyclase Heat shock protein 70-3 Larval cuticle protein LCP-17 Larval cuticle protein LCP-17 LCP18 Low molecular 30 kDa lipoprotein PBMHP-6 Malate dehydrogenase Mannosyltransferase Pyruvate carboxylase Scolexin Succinyl-CoA:3-ketoacid-coenzyme A transferase Superoxide dismutase [Cu-Zn] T-complex protein 1 subunit epsilon Transporter Troponin C 25D

Transmembrane Protein

Y

Y

Y

Y

Note: for “uncharacterized proteins”, refer to full list in Table S5. 5

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is a list of 37 insect O-glycan compositions that were used for database searching in this study based on literature reports (Kurz et al., 2015; Staudacher, 2015; Zhu et al., 2019). The O-glycoproteins identified in the silkworm strains 306 and NB (Table 3) were also N-glycosylated. In addition to the canonical O-glycans, silkworm was found to contains sulfate modifications, which has been reported for the dipteran species Anopheles and Drosophila (Kurz et al., 2015). Some proteins, including arylphorin and another silkworm storage protein, are found to be Ofucosylated with a single fucose residue, which is consistent with a previous study showing that both glycoproteins are O-glycosylated based on monosaccharide composition profiling (Kim et al., 2003). Our results confirm that O-glycosylation occurs at the single serine site S213 with heterogeneous glycan modification for arylphorin (Fig. 4), and at S210 and T594 for the other storage protein in both strains (Table 3). We also noted that arylphorin glycan heterogeneity is reduced in the NB strain, also the case for the myosin regulatory light chain (Table 3). However, there are possibilities that some atypical O-glycans may not be represented in the panel of glycans that were searched; for example, some may contain phosphocholine or methylation modifications in certain lepidopteran species (Stanton et al., 2017). Nevertheless, our results provided a preliminary O-glycosylation map in silkworm for the first time. As in the case of N-glycoproteins, most O-glycoproteins have not been characterized or even discovered, indicating a need for indepth investigations of the structure and function of these glycoproteins. During host-pathogen interactions, an important role of protein glycosylation on the host cell surface is to act as a targeted receptor for the pathogen. Our results provide insight into the relationship between host glycosylation and viral susceptibility. Variation in the O-glycoprotein profiles, or more specifically, a reduction in the O-glycan heterogeneity in proteins such as arylphorin and myosin regulatory light chain in the BmNPV resistant strain was observed. Similar findings have been reported for a mutant Caehorhabtidis elegans that was found to contain reduced O-glycan populations and is resistant to bacterial infections (Cipollo et al., 2004). The results indicate that a reduction in host glycosylation can potentially inhibit pathogen invasion.

Table 2 N-glycoproteins uniquely mapped in strain NB (partial). Protein ID

Mol. Weight (kDa)

Protein name

H9JWN1 P07837 H9J0S9 H9J1D2 H9IT95 H9JW98 H9IU42 H9J3R0 Q4F863 Q6T9Z7 Q2F5R4 H9J910 H9JM87 H9JSY6 H9JCE9 Q1HPS0 H9ITN9 H9JUN1 H9JLX1 C0H6L2 C0J8G3 H9JC47 H9JV50

41.8 41.8 40.0 44.3 45.8 63.3 62.6 61.8 17.5 38.1 61.4 84.0 48.2 46.6 105.9 22.0 16.6 28.7 62.8 35.9 45.8 19.4 50.0

Actin muscle-type A1 Actin muscle-type A2 Aspartate aminotransferase Aspartate aminotransferase Calreticulin Carboxylic ester hydrolase Carboxylic ester hydrolase Carboxylic ester hydrolase Eukaryotic translation initiation factor 5A Fibroinase Glutamate dehydrogenase Integrin beta Lipase Lipase Lon protease homolog mitochondrial Myosin regulatory light chain 2 Peptidylprolyl isomerase Phosphoglycerate mutase Polypeptide N-acetylgalactosaminyltransferase Putative cuticle protein Serpin-14 Transgelin Tubulin alpha chain

Note: for “uncharacterized proteins”, refer to full list in Table S6.

glycan compositional analysis and comparisons between the two species are needed in order to confirm this argument. 3.5. Silkworm contains O-glycoproteins that vary between the two strains To our best knowledge, there have been no O-glycosylated proteins reported for silkworm. The technical difficulties and minute quantities associated with this class of macromolecules account for the main obstacles in structural analysis. In this study, the glycopeptides were deaminated, which greatly reduced the heterogeneity raised by N-glycosylation, therefore, it was possible to map O-glycosylation by searching against a panel of insect O-glycan mass modifications to the serine or threonine residues in the enriched (glycol)peptides. Table S7 Table 3 Silkworm O-glycosylation profiles in strains 306 and NB. Uniprot ID

Protein name

Molecular weight (kDa)

306

NB

Sites

Compositions

Sites

Compositions

S213

Y

S210 T594 S403S S2373 S824

fucose,HexNAc, Hex1Pent, Hex1HexA1HexNAc, Hex1HexNAc2, HexNAc Hex2HexNAc2Sulf Hex2HexNAc HexNAc4 Hex1HexNAc2

S104

Hex2dHex2

Y

Q1HPP4

Arylphorin

83.4

S213

H9JHM9

Silkworm storage protein Uncharacterized protein Uncharacterized protein Myosin regulatory light chain 2 Uncharacterized protein Actin muscle-type A1 Uncharacterized protein Uncharacterized protein 30K protein 2 Uncharacterized protein Uncharacterized protein

82.8

S210 T594 T1120 S2373 S824

fucose,HexNAc, Hex1Pent, HexNAc2,Hex2dHex2, Hex2HexNAc,Hex1HexA1 HexNAc, Hex2HexNAc3,Hex2 HexNAc3Sulf fucose, HexNAc4 Hex2HexNAc3 Hex1HexNAc1 HexNAc4 Hex1HexNAc2

S104 S124 S273 S295

Hex2dHex2, fucose, Hex1HexNAc1dHex fucose Hex1HexNAc fucose

H9JRT0 H9JXG1 Q1HPS0 H9J9M0 H9JWN1 H9J4V7 H9J6I7 E5EVW2 H9J1X5 H9J7K1

425.7 123.6 22.0 61.4

Also Nglycosylated

Y Y Y Y Y

Y

41.8 789.0

T107 S2200

Hex1HexNAc Hex1HexNAc1Sulf

Y Y

227.9

S1181

fucose

Y

29.4 57.1

S34 T28

fucose fucose

Y Y

47.0

S292

HexNAc

Y

Abbreviation: Sulf: sulphation; Hex: hexose; HexNAc: N-acetylhexosamine; HexA: hexuronic acid; Fuc: fucose; Pent: pentose. 6

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Fig. 4. Peptide sequences and glycan site modifications of the mapped silkworm glycoproteins arylphorin from 306 (left) and NB (right). Different modifications are color coded.

4. Conclusion

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Silkworm glycobiology has rarely been a focus for sericulture research except for the production of mammalian type glycoproteins via baculovirus expression system. We produced a systematic glycosylation map for the model silkworm Bombyx mori, and demonstrated its outstanding glycosylation capacity. The N- and O-glycoproteins profiles were compared between two silkworm strains 306 and NB, which are susceptible and resistant, respectively, to the baculovirus BmNPV. Our results show that some glycoproteins are uniquely mapped only to one strain and, of those, the transmembrane glycoproteins are particularly interesting for investigation of their potential roles in viral infection. In addition, we reported O-glycoproteins for the silkworm species for the first time. Differences in glycan compositions were also noted between the susceptible strain and the resistant strain. As is now generally recognized that alterations in protein glycosylation profiles may be indicative of physiological or pathological changes in organisms, our study underlines the connection between host glycosylation and its antiviral activities. Funding This work was supported by the National Natural Science Foundation of China (31702186 and 31872425), Natural Science Foundation of Jiangsu Province China (BK20160509), and China Postdoctoral Science Foundation (2016M601725). Declaration of Competing Interest None. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jip.2019.107250. References Arai, I., Ohta, M., Suzuki, A., Tanaka, S., Yoshizawa, Y., Sato, R., 2013. Immunohistochemical analysis of the role of hemocytin in nodule formation in the larvae of the silkworm, Bombyx mori. J. Insect Sci. 13 (1). Bagdonaite, I., Wandall, H.H., 2018. Global aspects of viral glycosylation. Glycobiology 28 (7), 443–467. Barrows, B.D., Haslam, S.M., Bischof, L.J., Morris, H.R., Dell, A., Aroian, R.V., 2007. Resistance to Bacillus thuringiensis Toxin in Caenorhabditis elegans from Loss of Fucose. J. Biol. Chem. 282 (5), 3302–3311. Bown, D.P., Gatehouse, J.A., 2004. Characterization of a digestive carboxypeptidase from the insect pest corn earworm (Helicoverpa armigera) with novel specificity towards C-terminal glutamate residues. Eur. J. Biochem. 271 (10), 2000–2011. Cipollo, J.F., Awad, A.M., Costello, C.E., Hirschberg, C.B., 2004. srf-3, a Mutant of Caenorhabditis elegans, Resistant to Bacterial Infection and to Biofilm Binding, Is

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Bombyx mori. Biochem. Biophys. Res. Commun. 495 (1), 839–845. Zhu, F., Li, D., Chen, K., 2019. Structures and functions of invertebrate glycosylation. Open Biology 9 (1), 180232. Zielinska, Dorota F., Gnad, F., Schropp, K., Wiśniewski, Jacek R., Mann, M., 2012. Mapping N-glycosylation sites across seven evolutionarily distant species reveals a divergent substrate proteome despite a common core machinery. Mol. Cell 46 (4), 542–548. Zielinska, D.F., Gnad, F., Wiśniewski, J.R., Mann, M., 2010. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell 141 (5), 897–907.

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