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Identification of novel members reveals the structural and functional divergence of lepidopteran-specific Lipoprotein_11 family

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Abstract

30K proteins (30KPs) are classified into the lepidopteran-specific Lipoprotein_11 family. They are involved in various physiological processes such as energy storage, embryonic development, and immune response in the silkworm. To date, 30KPs were only found in Bombyx mori and Manduca sexta. Moreover, the C-termini of ENF peptide binding proteins (ENF-BPs) show similarity to 30KPs. ENF peptides are multifunctional insect cytokines and involved in growth regulation and defense reaction, whereas ENF-BPs act as active regulators of ENF peptides. In order to get insights into this gene family in Lepidoptera, we performed an extensive survey of lepidopteran-derived genome and EST datasets. We identified 73 30KP homologous genes in 12 lepidopteran species, of which 56 are novel members. The structural and phylogenetic analyses revealed that these genes could be classified into three groups: ENF-BP genes, typical 30KP genes, and serine/threonine-rich 30KP (S/T-rich 30KP) genes. The C-terminal regions are common to all the three subfamilies, but the N-termini are highly variable. We found a novel subfamily of Lipoprotein_11 and named it S/T-rich 30KP according to its exclusive S/T-rich domain in the N terminus. ENF-BP was also found to contain a special domain in the N terminus, which is homologous to Pp-0912 of Pseudomonas putida. Microarray data and semi-quantitative RT-PCR showed that the three groups have their respective temporal–spatial expression patterns. S/T-rich 30KP genes have enriched expression in the mature testis and might be involved in spermiogenesis or fertilization. Typical 30KP genes are expressed mainly in the fat body and integument at the larvae and pupae stages. ENF-BP genes are expressed predominantly in the hemocyte. The differential spatial–temporal expression profiles revealed the functional divergence of three Lipoprotein_11 subfamilies.

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Acknowledgments

This work was supported by the National Basic Research Program of China (no. 2012CB114600), the National Hi-Tech Research and Development Program of China (no. 2011AA100306), the National Natural Science Foundation of China (no. 31172157), and the Graduate Technological Innovation Foundation of Southwest University of China (no. kb2010003). We appreciate the valuable suggestions offered by the editor, and we also want to give our thanks to Dr. Hongjuan Cui for her careful reading of the manuscript.

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Correspondence to Ping Zhao.

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Supplementary Table 1

Primers used in semi-quantitative RT-PCR study. Primer sequences, melting temperatures, cycles, and amplicon sizes are listed. (DOC 72 kb)

Supplementary Table 2

Low molecular weight lipoproteins in the silkworm, B. mori. List of low molecular weight lipoproteins in the silkworm genome with the name, microarray probe, protein length, signal peptides, the location on the chromosome, and the EST expression evidence of each protein. Asterisk represents the location of the signal peptides cleavage site in the amino acid sequences of the low molecular weight lipoproteins. UN unknown chromosome locations. (DOC 118 kb)

Supplementary Table 3

Annotation of low molecular weight lipoproteins deposited in GenBank. List of the low molecular weight lipoproteins with accession number, definition, and the identities between predicted and reported low molecular weight lipoproteins. (DOC 39 kb)

Supplementary Table 4

List of the low molecular weight lipoproteins identified in lepidopteran insects except B. mori. Gene names are available from the ButterflyBase, WildSilkBase, etc. The best hit of silkworm low molecular weight lipoprotein genes with these genes are list. E value and the tissues which have EST evidences are also provided. (DOC 54 kb)

Supplementary Table 5

Synonymous and nonsynonymous substitutions of ENF-BP genes. A Synonymous and nonsynonymous substitutions across ENF-BP gene pairs. B Synonymous and nonsynonymous substitutions of the ENF-BP clusters. (DOC 45 kb)

Supplementary Fig. 1

Sequence alignment of the amino acid sequences of the lepidopteran low molecular weight lipoproteins. Alignments were performed using ClustalX 1.83 with default parameters and followed by shading with GeneDoc. Identical residues are shaded black, while similar residues are gray. P. putida homologous domain (PPD), signal peptide (SP), serine/threonine-rich domain (STD), all-α N-terminal domain (NTD), and all-β C-terminal domain (CTD) are mark out with black or gray horizontal columns of boxes. Secondary structure was predicted using Jpred online service tool. Purple cylinders represent α-helix; yellow arrows represent β-sheet. Blue triangles represent the hydrophobic cavity-forming residues. Hollow red stars represent the corresponding residues of Gly162 and Val215 in the first and second CTD repeat of Bmlp7, and the solid red star is the corresponding residues of Tyr266 in the third CTD repeat and is the putative sugar-binding residue (Yang et al. 2011). (PDF 7476 kb)

Supplementary Fig. 2

Serine/threonine phosphorylation sites in the STD. Serine/threonine kinase-specific phosphorylation sites that predicted by the GPS software are marked by the blue color. (PDF 486 kb)

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Zhang, Y., Dong, Z., Liu, S. et al. Identification of novel members reveals the structural and functional divergence of lepidopteran-specific Lipoprotein_11 family. Funct Integr Genomics 12, 705–715 (2012). https://doi.org/10.1007/s10142-012-0281-4

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