Genome-wide prediction of splice-modifying SNPs in human genes using a new analysis pipeline called AASsites.

BACKGROUND: Some single nucleotide polymorphisms (SNPs) are known to modify the risk of developing certain diseases or the reaction to drugs. Due to next generation sequencing methods the number of known human SNPs has grown. Not all SNPs lead to a modified protein, which may be the origin of a disease. Therefore, the recognition of functional SNPs is needed. Because most SNP annotation tools look for SNPs which lead to an amino acid exchange or a premature stop, we designed a new tool called AASsites which searches for SNPs which modify splicing. RESULTS: AASsites uses several gene prediction programs and open reading frame prediction to compare the wild type (wt) and the variant gene sequence. The results of the comparison are combined by a handmade rule system to classify a change in splicing as "likely, probable, unlikely". Having received good results from tests with SNPs known for changing the splicing pattern we checked 80,000 SNPs from the human genome which are located near splice sites for their ability to change the splicing pattern of the gene and hereby result in a different protein. We identified 301 "likely" and 985 "probable" classified SNPs with such characteristics. Within this set 33 SNPs are described in the ssSNP Target database to cause modified splicing. CONCLUSIONS: With AASsites single SNPs can be checked for those causing splice modifications. Screening 80,000 known human SNPs we detected about 1,200 SNPs which probably modify splicing. AASsites is available at http://genius.embnet.dkfz-heidelberg.de/menu/biounit/open-husar using any web browser.

Assay and heterologous expression in Pichia pastoris of plant cell wall type-II membrane anchored glycosyltransferases.

Two Arabidopsis xylosyltransferases, designated RGXT1 and RGXT2, were recently expressed in Baculovirus transfected insect cells and by use of the free sugar assay shown to catalyse transfer of D-xylose from UDP-alpha-D-xylose to L-fucose and derivatives hereof. We have now examined expression of RGXT1 and RGXT2 in Pichia pastoris and compared the two expression systems. Pichia transformants, expressing soluble, secreted forms of RGXT1 and RGXT2 with an N- or C-terminal Flag-tag, accumulated recombinant, hyper-glycosylated proteins at levels between 6 and 16 mg protein * L(-1) in the media fractions. When incubated with 0.5 M L-fucose and UDP-D-xylose all four RGXT1 and RGXT2 variants catalyzed transfer of D-xylose onto L-fucose with estimated turnover numbers between 0.15 and 0.3 sec(-1), thus demonstrating that a free C-terminus is not required for activity. N- and O-glycanase treatment resulted in deglycosylation of all four proteins, and this caused a loss of xylosyltransferase activity for the C-terminally but not the N-terminally Flag-tagged proteins. The RGXT1 and RGXT2 proteins displayed an absolute requirement for Mn(2+) and were active over a broad pH range. Simple dialysis of media fractions or purification on phenyl Sepharose columns increased enzyme activities 2-8 fold enabling direct verification of the product formed in crude assay mixtures using electrospray ionization mass spectrometry. Pichia expressed and dialysed RGXT variants yielded activities within the range 0.011 to 0.013 U (1 U = 1 nmol conversion of substrate * min(-1) * microl medium(-1)) similar to those of RGXT1 and RGXT2 expressed in Baculovirus transfected insect Sf9 cells. In summary, the data presented suggest that Pichia is an attractive host candidate for expression of plant glycosyltransferases.

Functional characterisation of a putative rhamnogalacturonan II specific xylosyltransferase.

An Arabidopsis thaliana gene, At1g56550, was expressed in Pichia pastoris and the recombinant protein was shown to catalyse transfer of D-xylose from UDP-alpha-D-xylose onto methyl alpha-L-fucoside. The product formed was shown by 1D and 2D 1H NMR spectroscopy to be Me alpha-D-Xyl-(1,3)-alpha-L-Fuc, which is identical to the proposed target structure in the A-chain of rhamnogalacturonan II. Chemically synthesized methyl L-fucosides derivatized by methyl groups on either the 2-, 3- or 4 position were tested as acceptor substrates but only methyl 4-O-methyl-alpha-L-fucopyranoside acted as an acceptor, although to a lesser extent than methyl alpha-L-fucoside. At1g56550 is suggested to encode a rhamnogalacturonan II specific xylosyltransferase.

Hydrophobic ligand binding properties of the human lipocalin apolipoprotein M.

Apolipoprotein M (apoM) is a plasma protein associated mainly with HDL. ApoM is suggested to be important for the formation of prebeta-HDL, but its mechanism of action is unknown. Homology modeling has suggested apoM to be a lipocalin. Lipocalins share a structurally conserved beta-barrel, which in many lipocalins bind hydrophobic ligands. The aim of this study was to test the ability of apoM to bind different hydrophobic substances. ApoM was produced both in Escherichia coli and in HEK 293 cells. Characterization of both variants with electrophoretic and immunological methods suggested apoM from E. coli to be correctly folded. Intrinsic tryptophan fluorescence of both apoM variants revealed that retinol, all-trans-retinoic acid, and 9-cis-retinoic acid bound (dissociation constant = 2-3 microM), whereas other tested substances (e.g., cholesterol, vitamin K, and arachidonic acid) did not. The intrinsic fluorescence of two apoM mutants carrying single tryptophans was quenched by retinol and retinoic acid to the same extent as wild-type apoM, indicating that the environment of both tryptophans was affected by the binding. In conclusion, the binding of retinol and retinoic acid supports the hypothesis that apoM is a lipocalin. The physiological relevance of this binding has yet to be elucidated.

Megalin is a receptor for apolipoprotein M, and kidney-specific megalin-deficiency confers urinary excretion of apolipoprotein M.

Apolipoprotein (apo) M is a novel apolipoprotein belonging to the lipocalin protein superfamily, i.e. proteins binding small lipophilic compounds. Like other apolipoproteins, it is expressed in hepatocytes and secreted into plasma where it associates with high-density lipoprotein particles. In addition, apoM is expressed at high levels in the kidney tubule cells. In this study, we show that the multiligand receptor megalin, which is expressed in kidney proximal tubule cells, is a receptor for apoM and mediates its uptake in the kidney. To examine apoM binding to megalin, a recombinant apoM was expressed in Escherichia coli and used in surface plasmon resonance and cell culture studies. The results showed apoM binding to immobilized megalin [dissociation constant (Kd) approximately 0.3-1 microm] and that the apoM was endocytosed by cultured rat yolk sac cells in a megalin-dependent manner. To examine the importance of apoM binding by megalin in vivo, we analyzed mice with a tissue-specific deficiency of megalin in the kidney. Megalin deficiency was associated with pronounced urinary excretion of apoM, whereas apoM was not detected in normal mouse, human, or rat urine. Gel filtration analysis showed that the urinary apoM-containing particles were small and devoid of apoA-I. The results suggest that apoM binds to megalin and that megalin-mediated endocytosis in kidney proximal tubules prevents apoM excretion in the urine.

Characterization of apoM in normal and genetically modified mice.

A novel human apolipoprotein [apolipoprotein M (apoM)] was recently described and demonstrated to be a lipocalin. We have now examined apoM in wild-type mice and mice with genetically altered lipoprotein metabolism. Liver and kidney showed high mRNA expression, whereas spleen, heart, brain, and testis demonstrated low expression. ApoM gene expression was initiated on embryonic day 10. Western blot analysis of plasma suggested that mouse apoM, like its human counterpart, is secreted with a retained signal peptide, but unlike human apoM it is not glycosylated. Gel filtration of plasma showed apoM to be associated with HDL-sized particles in wild-type and apoA-I-deficient mice and with HDL- and LDL-sized particles in LDL receptor-deficient mice, whereas apoM was mainly found in VLDL-sized particles in high-fat, high-cholesterol-fed apoE-deficient mice. The plasma concentration of apoM was similar in wild-type, LDL receptor-deficient, and apoE-deficient mice but was reduced to 33% in apoA-I-deficient compared with wild-type mice (P = 0.007). These data suggest that apoM mainly associates with HDL in normal mice but also with the pathologically increased lipoprotein fraction in genetically modified mice. The substantially decreased apoM levels in apoA-I-deficient mice suggest a connection between apoM and apoA-I metabolism.