The ribophorin I from Penaeus monodon shrimp: cDNA cloning, expression and phylogenetic analysis.
Ribophorin I, a 67 kDa subunit of the oligosaccharyl transferase complex, is involved in facilitating N-linked glycosylation of polypeptides. We have isolated a full length Penaeus monodon cDNA encoding an insect/mammalian ribophorin I homologue by screening a lymphoid cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction of lymphoid RNA. The cDNA clone of shrimp ribophorin I (PmRibI) consists of 2263 nucleotides encoding 601 amino acid residues. Primary structure analysis of PmRibI indicated that it is a type I transmembrane protein, comprising a cleavable signal sequence of 23 residues at the amino terminus, preceding 434 residues of the luminal domain, 17 residues of the transmembrane domain, and 150 residues of the cytoplasmic domain at the carboxy terminus. The protein has a calculated molecular mass of 67.98 kDa with a pI of 6.05. This putative PmRibI cDNA clone was also expressed as PmRibI-6His in Sf9 cells. The recombinant PmRibI has an apparent molecular weight of 70 kDa, similar to the MW calculated from the deduced cDNA sequence. The inferred protein sequence of PmRibI has 52% identity with that of Strongylocentrotus purpuratus, 49% identity with that of Danio rerio, and 47% identity with mammalian ribophorin I. Phylogenetic analysis showed that PmRibI is most closely related to the echinoderm ribophorin I. The expression of the ribophorin I gene is tissue specific, with its mRNA highly abundant in hemocytes, gill, lymphoid organ and hepatopancreas.
Chotwiwatthanakun
Charoonroj
C
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
Ngopon
Jutamas
J
Unajak
Sasimanas
S
Jitrapakdee
Sarawut
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D015870:Gene Expression; D008565:Membrane Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014018:Tissue Distribution
D018076:DNA, Complementary; D008565:Membrane Proteins; D011994:Recombinant Proteins; C034241:ribophorin