A cDNA clone encoding a new type of GalNAc alpha 2,6-sialyltransferase (ST6GalNAc II) with a structure similar to that of a previously cloned GalNAc alpha 2,6-sialyltransferase (ST6GalNAc I; Kurosawa, N., Hamamoto, T., Lee, Y.-C., Nakaoka, T., Kojima, N., and Tsuji, S. (1994) J. Biol. Chem. 269, 1402-1409) was obtained from chicken testes. The predicted amino acid sequence of ST6GalNAc II encodes a protein with type II transmembrane topology, as found for other glycosyltransferases, and showed 32% identity with that of ST6GalNAc I. Transfection of the full length ST6GalNAc II gene into COS cells led to GalNAc alpha 2,6-sialyltransferase activity with a different substrate specificity from that of ST6GalNAc I. Moreover, asialofetuin after treatment with beta-galactosidase did not serve as an acceptor for this enzyme. 14C-Sialylated oligosaccharides obtained from resialylated asialobovine submaxillary mucin with this enzyme were identical to Gal beta 1,3([14C]NeuAc alpha 2,6)GalNAc-ol but not [14C]NeuAc alpha 2,6GalNAc-ol. These results clearly show that the expressed enzyme is a novel type of sialyltransferase that requires beta-galactoside residues linked to GalNAc residues, whereas sialic acid residues linked to galactose residues are not essential for the activity.
The energy transduction in biomembranes requires a special supramolecular structure. This structure is needed for converting proton motive energy into ATP synthesis. These biomembranes contain 4 enzyme complexes that are composed of many polypeptides. Here we describe the following 4 phenomena : 1) oligomer formation by self-assembly of the subunits, 2) incorporation of the oligomers into a lipid bilayer, 3) chaperonin-mediated formation of the oligomers and membranes, 4) coordination of the subunit biosyntheses by enhancers and the role of mitochondrial DNA.Polypeptides translated are assembled into the membrane complexes with or without the help of chaperonins. In the case of thermophilic proteins, the reconstitutability of polypeptides is excellent ; for example, the catalytic portion of ATP synthase, α1 β1 subunit complex, was reconstituted. On the other hand, mesophilic ATP synthase requires chaperonins for its formation. The transcription of mRNAs for these polypeptides is coordinated by special enhancer and silencer. The role of polypeptides encoded by mitochondrial DNA in the molecular assembly was studied with the wild-type and mutant cells.
Based on the sequences of the highly conserved segments in the previously cloned sialyltransferases, a cDNA encoding a new type of Galp1,3GalNAc (u2,3-sialyltransferase (ST3GalA.2) has been isolated from both mouse and rat brain cDNA libraries.The cDNA sequences included an open reading frame coding for 350 amino acids, and the primary structure of this enzyme suggested a putative domain structure consisting of four regions, like that in other glycosyltransferases.The deduced amino acid sequence of ST3GalA.2 (mouse) showed 76% identity in the active domain with that of the previously cloned mouse Galpl,3GalNAc a2,3-sialyltransferase (ST3GalA.l(Lee, Y.-C., Kurosawa, N., Hamamoto, T., Nakaoka, T., and Tsuji, S. (1993) Eur.J. Biochern.216, 377-385)).Northern blotting indicated that the expression of ST3GalA.2 mRNA is tissue-specific, it being prominent in brain and liver, while that in the other tissues is very low.This enzyme expressed in COS-7 cells exhibited transferase activity only toward the disaccharide moiety of Galp1,SGalNAc of glycolipids as well as glycoproteins and oligosaccharides like ST3GalA.1, but showed a difference in acceptor substrate preference, Le. asialo-G,, and G,, were much more suitable substrates for ST3GalA.2 than for ST3GalA.l.The sialyltransferases comprise a family of glycosyltransferases that catalyze the transfer of sialic acid from CMP-NeuAc to the terminal position of the carbohydrate groups of glycoproteins and glycolipids (1, 2).It is not clear whether or not the sialyltransferases for glycoproteins are different from those for glycolipids.Precise molecular knowledge of sialyltransferases is vital for understanding the regulatory mechanism for the sialylation of glycoconjugates.So far, only a few sialyltransferases have been purified, which exhibit strict acceptor substrate specificities (3-6).In recent years, cDNAs encoding Galpl,4GlcNAc a2,6-sialyltransferases from rat liver
Purified alanine carrier proteins were cleaved into peptides either chemically after solubilization in 1,1,1,3,3,3-hexafluoro-2-propanol or proteolytically with lysylendopeptidase. From the amino acid sequence analyses of these peptides, we synthesized a DNA probe and utilized it for successful cloning of a gene encoding the alanine carrier protein (acp gene). The 5'-flanking region was determined by an inverse polymerase chain reaction, and an open reading frame consisting of 1,335 nucleotides was found. The amino acid sequence deduced from the open reading frame consists of 445 amino acids, and all the partial amino acid sequences determined are included in the sequence. Although the calculated M(r) of 47,803 is significantly larger than the apparent M(r) of 42,500 as reported previously (Hirata, H., Kambe, T., and Kagawa, Y. (1984) J. Biol. Chem. 259, 10653-10656), an in vitro translation experiment revealed that the product of the acp gene migrates at a position coinciding with that of the purified alanine carrier. Hydropathy analysis suggests that the protein contains at least 8 hydrophobic segments presumably spanning membrane. A homology search on a database reveals relatively high scores of homology with either the Escherichia coli melibiose carrier or the human Na+/glucose symporter, particularly in the region from Leu246 to Glu286. Furthermore, the region also reveals low but significant similarities to other Na(+)-coupled symporters.
