Characterization, Expression And Phylogenetic Analysis Of Amphioxus Cathepsin, β-MSP And Allantoicase Genes

Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. With its small, relatively unduplicated genomes, and lack of some key vertebrate innovations such as neural crest, ectodermal placodes and a cartilaginous or bony endoskeleton, amphioxus is the best available model having been widely used for interspecies comparative genome studies and developmental homology analysis. Researches on the characterization, expression and phylogenetic analysis of genes from amphioxus are increasing, but most of them were cloned from developing ectoderm or mesoderm or their derivatives. Information on the characterization, expression and phylogenetic analysis of endoderm-related genes is very much limited.Two cDNAs obtained from the endoderm derivatives -gut cDNA library of amphioxus B. belcheri tsingtauense is 1480 bp and 1700bp long. The longest open reading frames code for 327 amino acids and 333 amino acids with predicted molecular mass of approximately 36.3 KDa and 36.5 KDa, respectively. Of the two amino acids sequences, one sequence includes conserved motifs for cathepsin L proteinases, ERFNIN, and the other contains a conserved occluding loop, a unique feature of cathepsin B. These indicate that the cDNAs encode amphioxus cathepsin L and cathepsin B proteinases, and are thus designated AmphiCL and AmphiCB, respectively. Phylogenetic tree revealed that both AmphiCL and AmphiCB appear more closely related to invertebrate cathepsin L and cathepsin B, supporting the views that amphioxus is an animal transitional from invertebrates to vertebrates in phylogeny. Compared to 5 introns in the genome DNA of shrimp Penaeus cathepsin L and 7 introns in the genome DNAs of mammals' cathepsin Ls, the genome DNA of amphioxus cathepsin L with 2980bp long possesses 6 introns. Whether the number of introns in cathepsin L genome DNA is implicated in the evolution of cathepsin L remains further study. RT-PCR, Northern blot andsection in situ hybridization experiments showed that both AmphiCL and AmphiCB expressed in all tissues exmined, with the most abundance in the hepatic caecum and hind-gut, suggesting the role of food digestion for the two enzymes. Whole mount in situ hybridization analysis revealed that AmphiCL was firstly detected in the primitive gut of 2-day larvae, whereas AmphiCB was firstly detected in the primitive gut of 1-day larvae, suggesting that AmphiCB plays its physiological role in advance compared with AmphiCL. For further study of AmphiCL possible function, amphioxus was fed with spirulina Arthrospira platensis powder or treated with 1 ug/ml LPS. AmphiCL expression was down regulated in the guts of amphioxus when fed with spirulina and up regulated when exposed to LPS. AmphiCL may play a role in inflammatory reaction in amphioxus.P-microseminoprotein-like (fi-MSPL) is another gene cloned from the gut cDNA library of amphioxus. P-MSPL contains 111 amino acids with a predicted molecular mass of approximately 12.3 kDa. An alignment of P-MSPL with that of p-MSP family proteins revealed that P-MSPL shares only 12-20% similarity to the vertebrate P-MSP proteins. Comparison of the p-MSPL with P-MSP proteins of other vertebrates at the amino acid level showed that only P-MSPL doesn't contain a signal peptide at N-terminal and only eight cysteines are conserved, whereas in the vertebrate P-MSP proteins with ten cysteines conserved. These not only confirm the previous observation that P-MSP is a rapidly evolving protein during phylogeny, but also provide further data on the degree of diversity between species of this protein. Phylogenetic tree constructed using the amino acid sequence of amphioxus P-MSPL and that of other known P-MSPs showed that amphioxus P-MSPL is clubbed together with P-MSPs from various fish. Results of both RT-PCR and Northern blot showed that P-MSPL transcripts were detected in all tissues examined, with most abundance in the notochord, suggesting a possible role for notochord function. Whole mount in situ hybridization analysis revealed that positive hybridization signals were present in all blastomeres of the embryos from 4-cell to gastrula stages; In neural plate stage embryos, P-MSPL transcripts were restricted to the endomesoderm; In late neurula stage embryos (approximately 10 somite pairs), expression was found in the notochord, somites and primitive gut; In 1-day larvae, expression persisted in the notochord, somites and primitive gut. The initial presence and later disappearance of p-MSPL transcripts in the ectodermsuggest that 0-MSPL is possibly relevant to the differentiation of ectoderm during embryonic development of cephalochordate amphioxus. This is the first report on the developmental expression of 0-MSP/p-MSPL gene in all organisms.The last gene reported here obtained from the gut cDNA library of amphioxus is allantoicase. Amphioxus allantoicase contains 392 amino acids with a predicted molecular mass of 43.2 kDa. It includes all the conserved motifs characteristic of allantoicases, DGWET(S)RR, PDGG and DGWETAR. Amphioxus allantoicase is localized in cytoplasm with a 43.5% probability (only with a 21.7% probability of mitochondria] or peroxisomal localization) by PSORTII program anakysis. Analysis by the Protparam Tool program shows that amphioxus allantoicase appears an unstable enzyme, such the case in the zebrafish and frog. These suggest that the loss of allantoicase stability in purine metabolism occurred in cephalochordates. Phylogenetic tree demonstrated that amphioxus allantoicase was clustered together with that of zebrafish, suggesting amphioxus allantoicase is akin to vertebrate allantoicases. RT-PCR experiment showed that allantoicase gene was strongly expressed in the hepatic caecum, while weakly in other tissues including the hind-gut, gill, muscle, notochord, testis and ovary. A parallel experiment was performed measuring the allantoicase activity in the same tissues. It appears that in the hepatic caecum, allantoicase mRNA is more efficiently translated, while in other tissues examined, the enzymatic activity is relatively low or undetectable. Similar tissue specificity has also been found for amphibian allantoicase.