Molecular characterization, expression, cellular distribution and functional analysis of the shrimp (metapenaeus ensis) farnesoic acido-methyltransferase : a novel enzyme in the biosynthetic pathway of methyl farnesoate

by Gunawardene, Yasanthi Illika

(Uncorrected OCR) Abstract of thesis entitled MOLECULAR CHARACTERIZATION, EXPRESSION, CELLULAR DISTRIBUTION AND FUNCTIONAL ANALYSIS OF THE SHRIMP (Metapenaeus ensis) FARNESOIC ACID O-METHYLTRANSFERASE: A NOVEL ENZYME IN THE BIOSYNTHETIC PATHWAY OF METHYL FARNESOATE Submitted by Yasanthi Illika Nilmini Silva Gunawardene for the degree of Doctor of Philosophy at The University of Hong Kong in October 2002 Methyl farnesoate (MF) is implicated in the regulation of growth and reproduction of crustaceans. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the conversion of farnesoic acid (FA) to MF in the mandibular organ (MO). Recent studies also indicate the possible involvement of eyestalk neuropeptides in mediating MF synthesis through FAMeT. Thus a comprehensive molecular and biochemical analysis of FAMeT is imperative to better understand the regulation of MF biosynthesis and the role of MF in crustaceans. In this study, RT-PCR, library screening and 5RACE were used to clone the FAMeT cDNA from the shrimp, Metapenaeus ensis. The shrimp FAMeT cDNA is 1.95 Kb in size with the longest open reading frame of 843 bp. It encodes a protein of 280 amino acids with a predicted molecular weight of 31.94 kDa. FAMeT is encoded by a single uninterrupted gene and its transcripts are widely distributed in many tissues in the shrimp. Unlike lobsters and crabs, the expression level of FAMeT is low in the MO but high in the ventral nerve cord (VNC). In the VNC, FAMeT can be detected in both sexes during the molting and reproductive cycles. However, FAMeT is not detected in the eyestalk of juvenile females. To study the distribution of FAMeT in shrimp, recombinant FAMeT (rFAMeT) was produced in E. coli by an expression vector. The purified fusion protein was used for antibody generation. FAMeT can be detected in the VNC, heart, eyestalk, muscle, ovary, testis, epidermis and hepatopancreas. In agreement with the expression results, proteins immunoreactive to anti-rFAMeT serum were concentrated in the VNC. The results suggest that FAMeT in the VNC may remain inactive and may be transported to other tissues for enzymatic function. As demonstrated by a radiochemical bioassay, rFAMeT methylates FA to MF. Although the VNC consists of the highest concentration of FAMeT, the heart extract exhibited the highest FAMeT activity. Contrary to previous reports in other crustaceans, the MO extract shows only a low level of FAMeT activity in shrimp. The apparent co-localization of FAMeT and eyestalk neuropeptides in the X-organ sinus gland (XOSG) complex suggests a possible interaction between FAMeT and eyestalk neuropeptides. By Western blot analysis, a larger protein (~44 kDa) immunopositive to both anti-rCHHA and anti-rFAMeT serum was identified. Preliminary analysis of this large eyestalk protein suggests this protein may have antigenic determinants similar to the FAMeT and CHH-A. More extensive studies are needed to resolve this issue. In conclusion, molecular characterization and functional analysis were carried out on the gene encoding the shrimp FAMeT. Based on the results emanating from this study, the shrimp FAMeT may have a wide spectrum of action in many tissues that contribute to the regulation of MF synthesis and function of MF in shrimp.