Mutational analysis of human hydroxysteroid sulfotransferase SULT2B1 isoforms reveals that exon 1B of the SULT2B1 gene produces cholesterol sulfotransferase,whereas exon 1A yields pregnenolone sulfotransferase

As a result of an alternative exon 1, the gene for human hydroxysteroid sulfotransferase (SULTB1) encodes for two peptides differing only at their amino termini. The SULT2B1b isoform preferentially sulfonates cholesterol. Conversely, the SULT2B1a isoform avidly sulfonates pregnenolone but not cholesterol. The outstanding structural feature that distinguishes the SULT2B1 isoforms from the prototypical SULT2A1 isozyme is the presence of extended amino- and carboxyl-terminal ends in the former. Investigating the functional significance of this unique characteristic reveals that removal of 53 amino acids from the relatively long carboxyl-terminal end that is common to both SULT2B1 isoforms has no effect on the catalytic activity of either isoform. On the other hand, removal of 23 amino acids from the amino-terminal end that is unique to SULT2B1b results in loss of cholesterol sulfotransferase activity, whereas removal of 8 amino acids from the amino-terminal end that is unique to SULT2B1a has no effect on pregnenolone sulfotransferase activity. Deletion analysis along with site-directed mutagenesis of SULT2B1b reveal that the amino acid segment 19-23 residues from the amino terminus and particularly isoleucines at positions 21 and 23 are crucial for cholesterol catalysis. In the gene for SULT2B1, exon 1B encodes for only the unique amino-terminal region of SULT2B1b; however, exon 1A encodes for the unique amino-terminal end of SULT2B1a plus an additional 48 amino acids. Thus, if the gene for SULT2B1 employs exon 1B, cholesterol sulfotransferase is synthesized, whereas if exon 1A is used, pregnenolone sulfotransferase is produced.     

The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the HCO3- salvage transporter human Na+-HCO3- cotransport isoform 3

Cystic fibrosis transmembrane conductance regulator (CFTR) regulates both HCO(3)(-) secretion and HCO(3)(-) salvage in secretory epithelia. At least two luminal transporters mediate HCO(3)(-) salvage, the Na(+)/H(+) exchanger (NHE3) and the Na(+)-HCO(3)(-) cotransport (NBC3). In a previous work, we show that CFTR interacts with NHE3 to regulate its activity (Ahn, W., Kim, K. W., Lee, J. A., Kim, J. Y., Choi, J. Y., Moe, O. M., Milgram, S. L., Muallem, S., and Lee, M. G. (2001) J. Biol. Chem. 276, 17236-17243). In this work, we report that transient or stable expression of human NBC3 (hNBC3) in HEK cells resulted in a Na(+)-dependent, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid)- and 5-ethylisopropylamiloride-insensitive HCO(3)(-) transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO(3)(-)-transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO(3)(-) transport by regulating the activity of all luminal HCO(3)(-) secretion and salvage mechanisms of secretory epithelial cells.     

The Hexapeptide inhibitor of Galbeta 1,3GalNAc-specific alpha 2,3-sialyltransferase as a generic inhibitor of sialyltransferases

The mammalian Galbeta1,3GalNAc-specific alpha2,3-sialyltransferase (ST3Gal I) was expressed as a secreted glycoprotein in High Five (Trichoplusia ni) cells. Using this recombinant ST3Gal I, we screened the synthetic hexapeptide combinatorial library to explore a sialyltransferase inhibitor. We found that the hexapeptide, NH(2)-GNWWWW, exhibited the most strong inhibition of ST3Gal I among five different hexapeptides that were finally selected. The kinetic analysis of ST3Gal I inhibition demonstrated that this hexapeptide could act as a competitive inhibitor (K(i) = 1.1 microm) on CMP-NeuAc binding to the enzyme. Moreover, the hexapeptide was shown to strongly inhibit both N-glycan-specific alpha2,3- and alpha2,6-sialyltranferase in vitro, suggesting that this peptide may inhibit the broad range of sialyltransferases regardless of their linkage specificity. The inhibitory activity in vivo was investigated by RCA-I lectin blot analyses and by metabolic d-[6-(3)H]GlcNH(2) radiolabeling analyses of N- and O-linked oligosaccharides in Chines hamster ovary cells. Our results demonstrate that the hexapeptide can act as a generic inhibitor of the N- and O-glycan-specific sialyltransferases in mammalian cells, which results in the significantly reduced NeuAc expression on cellular glycoproteins in vivo.     

The binding of Xanthophylls to the bulk light-harvesting complex of photosystem II of higher plants. A specific requirement for carotenoids with a 3-hydroxy-beta-end group

The pigment composition of the light-harvesting complexes (LHCs) of higher plants is highly conserved. The bulk complex (LHCIIb) binds three xanthophyll molecules in combination with chlorophyll (Chl) a and b. The structural requirements for binding xanthophylls to LHCIIb have been examined using an in vitro reconstitution procedure. Reassembly of the monomeric recombinant LHCIIb was performed using a wide range of native and nonnative xanthophylls, and a specific requirement for the presence of a hydroxy group at C-3 on a single beta-end group was identified. The presence of additional substituents (e.g. at C-4) did not interfere with xanthophyll binding, but they could not, on their own, support reassembly. cis isomers of zeaxanthin, violaxanthin, and lutein were not bound, whereas all-trans-neoxanthin and different chiral forms of lutein and zeaxanthin were incorporated into the complex. The C-3 and C-3' diols lactucaxanthin (a carotenoid native to many plant LHCs) and eschscholtzxanthin (a retro-carotenoid) both behaved very differently from lutein and zeaxanthin in that they would not support complex reassembly when used alone. Lactucaxanthin could, however, be bound when lutein was also present, and it showed a high affinity for xanthophyll binding site N1. In the presence of lutein, lactucaxanthin was readily bound to at least one lutein-binding site, suggesting that the ability to bind to the complex and initiate protein folding may be dependent on different structural features of the carotenoid molecule. The importance of carotenoid end group structure and ring-to-chain conformation around the C-6-C-7 torsion angle of the carotenoid molecule in binding and complex reassembly is discussed.     

Regulator of G protein signaling Z1 (RGSZ1) interacts with Galpha i subunits and regulates Galpha i-mediated cell signaling

Regulator of G protein signaling (RGS) proteins constitute a family of over 20 proteins that negatively regulate heterotrimeric G protein-coupled receptor signaling pathways by enhancing endogenous GTPase activities of G protein alpha subunits. RGSZ1, one of the RGS proteins specifically localized to the brain, has been cloned previously and described as a selective GTPase accelerating protein for Galpha(z) subunit. Here, we employed several methods to provide new evidence that RGSZ1 interacts not only with Galpha(z,) but also with Galpha(i), as supported by in vitro binding assays and functional studies. Using glutathione S-transferase fusion protein pull-down assays, glutathione S-transferase-RGSZ1 protein was shown to bind (35)S-labeled Galpha(i1) protein in an AlF(4)(-)dependent manner. The interaction between RGSZ1 and Galpha(i) was confirmed further by co-immunoprecipitation studies and yeast two-hybrid experiments using a quantitative luciferase reporter gene. Extending these observations to functional studies, RGSZ1 accelerated endogenous GTPase activity of Galpha(i1) in single-turnover GTPase assays. Human RGSZ1 functionally regulated GPA1 (a yeast Galpha(i)-like protein)-mediated yeast pheromone response when expressed in a SST2 (yeast RGS protein) knockout strain. In PC12 cells, transfected RGSZ1 blocked mitogen-activated protein kinase activity induced by UK14304, an alpha(2)-adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2 dopamine receptor agonist-induced serum response element reporter gene activity in Chinese hamster ovary cells. In summary, these data suggest that RGSZ1 serves as a GTPase accelerating protein for Galpha(i) and regulates Galpha(i)-mediated signaling, thus expanding the potential role of RGSZ1 in G protein-mediated cellular activities.     

Molecular diversity of arbuscular mycorrhizal fungi and patterns of host association over time and space in a tropical forest

We have used molecular techniques to investigate the diversity and distribution of the arbuscular mycorrhizal (AM) fungi colonizing tree seedling roots in the tropical forest on Barro Colorado Island (BCI), Republic of Panama. In the first year, we sampled newly emergent seedlings of the understory treelet Faramea occidentalis and the canopy emergent Tetragastris panamensis, from mixed seedling carpets at each of two sites. The following year we sampled surviving seedlings from these cohorts. The roots of 48 plants were analysed using AM fungal-specific primers to amplify and clone partial small subunit (SSU) ribosomal RNA gene sequences. Over 1300 clones were screened for random fragment length polymorphism (RFLP) variation and 7% of these were sequenced. Compared with AM fungal communities sampled from temperate habitats using the same method, the overall diversity was high, with a total of 30 AM fungal types identified. Seventeen of these types have not been recorded previously, with the remainder being similar to types reported from temperate habitats. The tropical mycorrhizal population showed significant spatial heterogeneity and nonrandom associations with the different hosts. Moreover there was a strong shift in the mycorrhizal communities over time. AM fungal types that were dominant in the newly germinated seedlings were almost entirely replaced by previously rare types in the surviving seedlings the following year. The high diversity and huge variation detected across time points, sites and hosts, implies that the AM fungal types are ecologically distinct and thus may have the potential to influence recruitment and host composition in tropical forests.