Apolipoprotein M (apoM) is a plasma protein associated mainly with HDL.

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-Ideficient 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-cholesterolfed 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.

An Arabidopsis thaliana gene, At1g56550, was expressed in Pichia pastoris and the recombinant protein was shown to catalyse transfer of D D-xylose from UDP-a-D D-xylose onto methyl a-L L-fucoside. The product formed was shown by 1D and 2D 1 H NMR spectroscopy to be Me a-which is identical to the proposed target structure in the A-chain of rhamnogalacturonan II. Chemically synthesized methyl L 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-a-L L-fucopyranoside acted as an acceptor, although to a lesser extent than methyl a-L L-fucoside. At1g56550 is suggested to encode a rhamnogalacturonan II specific xylosyltransferase.

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.

BackgroundSome 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.ResultsAASsites 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.ConclusionsWith 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.