MsVmp1 is extensively <i>O</i>-glycosylated.

<p>(A) <i>MsVMP1</i> was expressed in insect Sf21 cells and purified by Ni-NTA chromatography. After SDS-PAGE, the proteins were transferred to a nitrocellulose membrane and stained with Ponceau Red (left) and subsequently with different lectins (right lanes). GNA, <i>Galanthus nivalis</i> agglutinin; PNA, peanut agglutinin; SNA, <i>Sambucus nigra</i> agglutinin; DSA, <i>Datura stramonium</i> agglutinin; MAA, <i>Maackia amurensis</i> agglutinin. (B) For deglycosylation, MsVmp1 (expressed in insect cells) was treated with PNGase F or <i>O</i>-glycosidase in the presence of protease inhibitors. The reaction products were separated by SDS PAGE and stained with Coomassie Blue. <i>Left </i><i>lane</i>, MsVmp1 input without addition of glycosidase; Middle lane, PNGase F treatment; Right lane, <i>O</i>-glycosidase treatment. Std, standard proteins with indicated molecular masses in kDa. Arrows point to reaction intermediates and the terminal deglycosylation product with indicated molecular masses.</p

Immunoblotting detects MsVmp1 in the midgut of feeding, starving and molting larvae.

<p>Crude protein extracts from the anterior, median and posterior midgut were separated by SDS-PAGE, blotted onto nitrocellulose and stained with anti-VMP1 antibodies. The given molecular mass was estimated using standard proteins of known molecular masses. </p

ClustalW alignment of valine-rich midgut proteins from <i>M. sexta</i> (MsVmps).

<p>Highly conserved or identical amino acids are highlighted with light grey, grey or black shadings. The consensus sequence is given below. The accession numbers are as follows: MsVmp1 (Msex012254-PA), MsVmp2 (central part of Msex012257-PA), MsVmp3 (C-terminal part of Msex012257-PA), MsVmp4 (N-terminal part of Msex012261-PA), MsVmp5 (N-terminal part of Msex012257-PA), MsVmp6 (Msex011100-PA), MsVmp7 (C-terminal part of Msex012261-PA), MsVmp8 (Msex012260-PA), and MsVmp (Msex012262-PA).</p

Tissue specific expression of <i>MsVMP</i> genes in fifth instar larvae of <i>M. sexta</i>.

<p>Total RNA was prepared from various tissues and cDNAs were synthesized. RT-PCR was carried out with primers specific to the indicated genes. PCR products of indicated sizes were separated by agarose gel electrophoresis and stained with ethidium bromide. Products for the ribosomal protein MsRpS3 were used as a loading control. Expression was found exclusively in the midgut. </p

Immunodetection of MsVmp1 in the posterior midgut of <i>M. sexta</i> larvae at different physiological conditions.

<p>Cryosections of posterior midguts were stained with CFW and immune-labeled with anti-VMP1 antibodies. Primary antibodies were detected with ALEXA 488-conjugated anti-guinea pig IgGs. Brightfield and fluorescence images (for anti-Vmp1 and CFW), and overlays of fluorescence images are shown. Cryosections were obtained from feeding 2^nd instar larvae (top), starving 2^nd instar larvae (middle) and larvae molting from the 2^nd to the 3^rd instar. C, cuticle; EC, ectoperitrophic space; EN, endoperitrophic space; PM, peritrophic matrix. Size bar, 100 µm.</p

Immunodetection of MsVmp1 in PM preparations from anterior and posterior midguts of feeding larvae.

<p>(A) PM proteins were extracted by SDS treatment and separated by SDS-PAGE. Then the proteins were transferred to nitrocellulose and reacted with polyclonal anti-Vmp1 antibodies. Std, standard proteins with molecular masses indicated in kDa. (B) Immunodetection of Vmps using anti-Vmp1 antibodies. The PM preparations from the anterior and posterior parts of the midgut were washed several times with PBS buffer, blocked with bovine serum albumin and stained with the anti-Vmp1 antibodies. Cy3-conjugated anti-guinea pig IgGs were used as secondary antibodies. The PM was transferred to a microscope slide and mounted with Vectashield under a cover slip. The specimens were viewed under a fluorescence microscope using appropriate excitation an emission filters.</p

Phylogenetic tree of lepidopteran VMPs.

<p>The un-rooted maximum likelihood tree was calculated on the basis of a ClustalW alignment of VMPs from different lepidopteran species. Bootstrap values are given in percentages at the internodes; Accession numbers refer to the entries of the dbEST database of Butterflybase. MS, <i>Manduca sexta</i>; BM, <i>Bombyx mori</i>; TN, <i>Trichopulsia </i><i>ni</i>; HA, <i>Helicoverpa armigera</i>; SF, <i>Spodoptera frugiperda</i>; PI, <i>Plodia interpunctella</i>.</p

A lepidopteran-specific gene family encoding valine-rich midgut proteins

Citation: Odman-Naresh J, Duevel M, Muthukrishnan S, Merzendorfer H (2013) A Lepidopteran-Specific Gene Family Encoding Valine-Rich Midgut Proteins . PLOS ONE 8(11): e82015. https://doi.org/10.1371/journal.pone.0082015Many lepidopteran larvae are serious agricultural pests due to their feeding activity. Digestion of the plant diet occurs mainly in the midgut and is facilitated by the peritrophic matrix (PM), an extracellular sac-like structure, which lines the midgut epithelium and creates different digestive compartments. The PM is attracting increasing attention to control lepidopteran pests by interfering with this vital function. To identify novel PM components and thus potential targets for insecticides, we performed an immunoscreening with anti-PM antibodies using an expression library representing the larval midgut transcriptome of the tobacco hornworm, Manduca sexta. We identified three cDNAs encoding valine-rich midgut proteins of M. sexta (MsVmps), which appear to be loosely associated with the PM. They are members of a lepidopteran-specific family of nine VMP genes, which are exclusively expressed in larval stages in M. sexta. Most of the MsVMP transcripts are detected in the posterior midgut, with the highest levels observed for MsVMP1. To obtain further insight into Vmp function, we expressed MsVMP1 in insect cells and purified the recombinant protein. Lectin staining and glycosidase treatment indicated that MsVmp1 is highly O-glycosylated. In line with results from qPCR, immunoblots revealed that MsVmp1 amounts are highest in feeding larvae, while MsVmp1 is undetectable in starving and molting larvae. Finally using immunocytochemistry, we demonstrated that MsVmp1 localizes to the cytosol of columnar cells, which secrete MsVmp1 into the ectoperitrophic space in feeding larvae. In starving and molting larvae, MsVmp1 is found in the gut lumen, suggesting that the PM has increased its permeability. The present study demonstrates that lepidopteran species including many agricultural pests have evolved a set of unique proteins that are not found in any other taxon and thus may reflect an important adaptation in the highly specialized lepidopteran digestive tract facing particular immune challenges