Proteomic Identification of Bombyx mori Organelles Using the Engineered Ascorbate Peroxidase APEX and Development of Silkworm Organelle Proteome Database (SilkOrganPDB).

Tian Li,Chunfeng Li,Xianzhi Meng,Zeyang Zhou,Jian Luo,Jinzhi Xu,Chen Xu,Bin Yu,Guoqing Pan

doi:10.3390/ijms22095051

Abstract

Silkworm Bombyx mori is an economically important insect and a lepidopteran model. Organelle proteome is vital to understanding gene functions; however, it remains to be identified in silkworm. Here, using the engineered ascorbate peroxidase APEX, we constructed transgenic B. mori embryo cells (BmE) expressing APEX-NLS, COX4-APEX, APEX-Rev, and APEX-KDEL in nucleus, mitochondrial matrix (MM), cytosol, and endoplasmic reticulum (ER), and isolated the biotin-labeled proteins using streptavidin-affinity purification, respectively. The isolated proteins were determined using LC-MS/MS and annotated by searching B. mori genomes downloaded from GenBank, SilkBase, SilkDB 2.0, and SilkDB 3.0, resulting in 842, 495, 311, and 445 organelle proteins identified, respectively. We mapped the 296 MM proteins annotated in the GenBank data to mitochondrial protein databases of the fly, human, and mouse, and found that 140 (47%) proteins are homologous to 80 fly proteins, and 65 (22%) proteins match to 31 and 29 human and mouse proteins, respectively. Protein orthology was predicted in multiple insects using OrthoMCL, producing 460 families containing 839 proteins we identified. Out of 460 families, 363 were highly conserved and found in all insects, leaving only three proteins without orthology in other insects, indicating that the identified proteins are highly conserved and probably play important roles in insects. A gene ontology enrichment analysis by clusterProfiler revealed that the nucleus proteins significantly enriched in cellular component terms of nucleus and nucleolus, the MM proteins markedly enriched in molecular function terms of nucleotide binding, and the cytosol proteins mainly enriched in biological process terms of small molecule metabolism. To facilitate the usage and analysis of our data, we developed an open-access database, Silkworm Organelle Proteome Database (SilkOrganPDB), which provides multiple modules for searching, browsing, downloading, and analyzing these proteins, including BLAST, HMMER, Organelle Proteins, Protein Locations, Sequences, Gene Ontology, Homologs, and Phylogeny. In summary, our work revealed the protein composition of silkworm BmE organelles and provided a database resource helpful for understanding the functions and evolution of these proteins.

Highlights

Introduction distributed under the terms andSilk moth is a large group of lepidopteran insects that includes the domestic silkworm Bombyx mori and some wild animals such as Actias selene, Antheraea assamensis, Antheraea pernyi, Bombyx huttoni, Bombyx mandarina, Caligula japonica, Eriogyna pyretoum, Philosamia cynthiaricini, and Samia cynthia
Transgenic B. mori embryo cells (BmE) cells expressing APEX fused with a leading peptide including COX4, nuclear localization signal (NLS), Rev, and KDEL were made for translocating the APEX in mitochondrial matrix (MM), nucleus, cytosol, and endoplasmic reticulum (ER), respectively
The COX4-APEX was colocalized with mitochondria stained by MitoTracker Deep Red (Figure 2A), indicating that the COX4 led the APEX into the mitochondria

Summary

Introduction

Introduction distributed under the terms andSilk moth is a large group of lepidopteran insects that includes the domestic silkworm Bombyx mori and some wild animals such as Actias selene, Antheraea assamensis, Antheraea pernyi, Bombyx huttoni, Bombyx mandarina, Caligula japonica, Eriogyna pyretoum, Philosamia cynthiaricini, and Samia cynthia. Three silkworm genomes from the B. huttoni, B. mandarina, and B. mori have been sequenced and deposited in the GenBank database under the accession numbers GCA_002197625.1, GCA_003987935.1, and GCA_000151625.1, respectively. The genomic and genetic maps greatly promoted studies of the functional and comparative genomics of silkworms [1,2,3], especially with a high-quality genome assembly of B. mori obtained and published recently [4]. This improved assembly and annotation provided a more accurate reference for transcriptomics and proteomics studies. Little is known about the protein composition of silkworm subcellular organelles like the nucleus, mitochondria, cytosol, and endoplasmic reticulum (ER), except that Wang et al

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Conclusion