Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis (Microsporidia).
Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick spore wall composed of a glycoprotein-rich outer layer or exospore and a chitin-rich inner layer or endospore. In this study performed on the silkworm pathogen Nosema bombycis, we analyzed the spore wall proteins (SWPs) by proteomic-based approaches, MALDI-TOF MS and LC-MS/MS, and 14 hypothetical spore wall proteins (HSWPs) or peptides were obtained in total. Furthermore, we have examined the SWPs by SDS-PAGE and three main spore wall peptides were detected with molecular weights of 32.7 kDa (SWP32), 30.4 kDa (SWP30), and 25.3 kDa (SWP25), respectively. By N-terminal amino acid residue sequencing, and searching the genomic DNA shotgun database of N. bombycis, the complete ORFs of SWP30 and SWP32 were obtained, which encode for a 278- and a 316-amino acid peptide, respectively. Mouse polyclonal antibodies were raised against SWP30 and SWP32 recombinant proteins produced in Escherichia coli, and the results of indirect immunofluorescence assay (IFA) and immunoelectron microscopy (IEM) analyses indicated SWP30 to be an endosporal protein while SWP32 was shown to be an exosporal protein. Both SWP30 and SWP32 are included in the 14 HSWPs identified by MS, confirming the results of the proteomic-based approaches.
The Key Sericultural Laboratory of Agricultural Ministry, Southwest University, Chongqing, P. R. China.
Zhengli
Z
Wu
Yanhong
Y
Li
Guoqing
G
Pan
Xiaohui
X
Tan
Junhua
J
Hu
Zeyang
Z
Zhou
Zhonghuai
Z
Xiang
D000595:Amino Acid Sequence; D000818:Animals; D000908:Antibodies, Fungal; D001483:Base Sequence; D012831:Bombyx / Q000648:ultrastructure; D002473:Cell Wall / Q000737:chemistry*; D003001:Cloning, Molecular; D018388:Codon, Terminator; D016208:Databases, Factual; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D016881:Microsporidiosis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016818:Nosema / Q000166:cytology* / Q000302:isolation & purification; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000737:chemistry; D021382:Protein Sorting Signals; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013172:Spores, Fungal / Q000737:chemistry*
D000908:Antibodies, Fungal; D018388:Codon, Terminator; D005656:Fungal Proteins; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins