Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae.
A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation.
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Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05513-970 São Paulo, Brasil.
Cristofoletti
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PT
Pimenta
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DC
Ribeiro
A F
AF
Terra
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WR
Ferreira
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C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005243:Feces / Q000737:chemistry; D041981:Gastrointestinal Tract / Q000378:metabolism; D015870:Gene Expression; D007150:Immunohistochemistry; D019476:Insect Proteins / Q000737:chemistry / Q000502:physiology*; D007814:Larva / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D013712:Tenebrio / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D019476:Insect Proteins