Identification And Characterization Of Two Novel Rab Proteins

Dendritic cell (DC ) is one of the focuses of the immunology study because DC is the most potent antigen-presenting cells in the immune system. The search of novel molecules of DC will not only ease the study of antigen presenting and immune regulation but also help the exploration of some biological mechanisms. Large-scale random sequencing of the eDNA library of DC will yield novel molecules and the investigation of the function of them will make it easy for people to reconsider some life phenomena. hRabJ (human Rab protein with J domain) in this article was from the human DC cDNA library and mRabJ (mouse Rab protein with J domain) was the homologue of the hRabJ from mouse testis. mRabJ and hRabJ were similar to each other both in the structural and biological function aspects. Rab proteins are a large subfamily of the Ras superfamily that involves many kinds of GTP-binding proteins. Molecular weight of Rab ranges from 22 to 25 KD. It is believed now that Rab functions as a regulator of vesicle docking and fusion processes by switching between a GTP-binding form and a GDP-binding form and that Rab plays important roles in protein transport including endocytosis and exocytosis. The investigation of novel Rab proteins will cast new light on the study of the protein transport. EST (end sequence tag) of RabJ was first discovered in the process of large-scale sequencing of the human DC cDNA library and the full length of RabJ cDNA was obtained by 5扲ACE (rapid amplification of cDNA end). By using human DC eDNA as template we cloned the encoding region of RabJ which was used afterwards in the construction of hRabJ-pGEX-2T vector for GST fusion protein expression and pcDNA3.1- hRabj, nGFP-hRabJ and hRabJ-cGFP vectors for eukaryotic expression. Full-length hRabJ cDNA contained 1787 bp and an open reading frame (ORF) of 822bp which was predicted to encode a protein of 273 amino acids. Analysis of the predicted protein sequence of RabJ showed that it shared character of two protein families. Three guanine binding motifs and three phosphate/magnesium binding motifs, which were characteristic of the Rab proteins, were found at its N-terminal. And a conserved J domain including one HPD sequence was detected at its carboxyl tennini, which was shared by the other DnaJ/Hsp4O cochaperones and was implicated in the interaction of DnaJfHsp4O with Hsp7O proteins. Thus we named it as hRabJ (human Rab protein with J domain). Northern blot analysis of various tissues showed that RabJ was specifically expressed in testis with weak expression in ovary and brain, which may indicate its function in the reproduction process. In the various cell lines examined, weak expression was observed. RT-PCR results also were consistent with the northern blot results. The mRabJ homologue was cloned by using mouse testis eDNA as template and primers of hRabJ as primers, the predicted protein sequence of which had a similarity as high as 97% to hRabJ. Northern blot analysis of various mouse tissues showed that mRabJ was also predominantly expressed in testis with weak expression in heart, brain and skeletal muscle. Then we went on to study the biological function of both novel molecules. At first we utilized the proearyotic GST fusion protein expression system to purify the active GST-hRabJ protein in BL2 1 E. Co/i. and then biochemically investigate the nature of hRabJ. By using a -32P labeled GTP as a marker we explored the GTP binding activity and GTPase activity of hRabJ. Results showed that hRabJ could specifically bind with GTP and not w...