Gene Ontology annotations and resources.

The Gene Ontology (GO) Consortium (GOC, http://www.geneontology.org) is a community-based bioinformatics resource that classifies gene product function through the use of structured, controlled vocabularies. Over the past year, the GOC has implemented several processes to increase the quantity, quality and specificity of GO annotations. First, the number of manual, literature-based annotations has grown at an increasing rate. Second, as a result of a new 'phylogenetic annotation' process, manually reviewed, homology-based annotations are becoming available for a broad range of species. Third, the quality of GO annotations has been improved through a streamlined process for, and automated quality checks of, GO annotations deposited by different annotation groups. Fourth, the consistency and correctness of the ontology itself has increased by using automated reasoning tools. Finally, the GO has been expanded not only to cover new areas of biology through focused interaction with experts, but also to capture greater specificity in all areas of the ontology using tools for adding new combinatorial terms. The GOC works closely with other ontology developers to support integrated use of terminologies. The GOC supports its user community through the use of e-mail lists, social media and web-based resources.

Binding of human urokinase-type plasminogen activator to its receptor: residues involved in species specificity and binding.

Urokinase-type plasminogen activator (UPA), particularly when bound to its receptor (UPAR), is thought to play a major role in local proteolytic processes, thus facilitating cell migration as may occur during angiogenesis, neointima and atherosclerotic plaque formation, and tumor cell invasion. To facilitate understanding of the need and function of the UPA/UPAR interaction in cell migration and vascular remodeling, we changed several amino acid residues in UPA so as to interfere with its interaction with its receptor. The receptor-binding domain of UPA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of UPA to UPAR appears to be species specific, we used the differences in amino acid sequences in the growth factor domain of UPA between various species to construct a human UPA variant that does not bind to the human UPAR. We substituted Asn22 for its mouse equivalent Tyr by site-directed mutagenesis. This mutant UPA had similar plasminogen activator characteristics as wild-type UPA, including its specific activity and interaction with plasminogen activator inhibitor-1. However, no UPA/UPAR complexes could be observed in cross-linking experiments using DFP-treated 125I-labeled mutant UPA and lysates of various cells, including U937 histiocytic lymphoma cells, phorbol myristate acetate-treated human ECs, and mouse LB6 cells transfected with human UPAR cDNA. In direct binding experiments, DFP-treated 125I-labeled mutant UPA could not bind to phorbol myristate acetate-treated ECs, whereas wild-type UPA did bind. Furthermore, a 25-fold excess of wild-type UPA completely prevented the binding of DFP-treated 125I-labeled wild-type UPA to the human receptor on transfected LB6 cells, whereas an equal amount of mutant UPA had only a very small effect. In ligand blotting assays, very weak binding of mutant UPA to human UPAR could be observed. Changing Asn22 into the other amino acid residues alanine or glutamine had no effect on binding to UPAR on human ECs. The functional integrity of the growth factor domain in the non-receptor binding Asn22Tyr mutant is suggested by the fact that binding of this mutant to a murine UPAR can be restored after additional mutations in the growth factor domain, Asn27,His29,Trp30 to Arg27,Arg29,Arg30. We conclude that Asn22 and Asn27,His29,Trp30 in human UPA are key determinants in the species-specific binding of the enzyme to its receptor and that changing Asn22 into Tyr results in a UPA mutant with strongly reduced binding to UPAR.

Identification of a distinct human high-density lipoprotein subspecies defined by a lipoprotein-associated protein, K-45. Identity of K-45 with paraoxonase.

In an attempt to provide immunological tools for subfractionation of high-density lipoproteins (HDL), monoclonal antibodies were raised against HDL complexes. Two clones identified a peptide, provisionally named K-45 (pI 4.5-4.9; molecular mass 45 kDa, range 42-48 kDa), whose plasma distribution and lipoprotein association were fully characterised. Gel filtration localised the peptide to the HDL region of human plasma where it co-eluted with apolipoprotein (apo) A-I, the structural protein of HDL. Complementary studies employing immunoabsorption with anti-(apo A-I) antibodies removed 90% of K-45 from plasma: conversely, anti-(apo A-II) antibodies eliminated only 10% of K-45. Immunoaffinity chromatography on an anti-(K-45) column revealed that the peptide was present in a distinct HDL subsepecies containing three major proteins: K-45, apo A-I and clusterin or apo J. The lipoprotein nature of the bound fraction was indicated by electron microscopy (diameter 9.6 +/- 3.3 nm) and quantification of lipids, the latter showing an unusually high triacyglycerol concentration. Plasma concentrations of K-45 were positively correlated with apo A-I and HDL-cholesterol and negatively correlated with apo B and total cholesterol. Thus, the peptide appears to be linked, directly or indirectly, to processes which give rise to an anti-atherogenic lipid profile. After completion of the present studies, an N-terminal sequence identical to that of K-45 was reported in recently isolated cDNA clones. These clones encode paraoxonase.

Distribution of apolipoprotein E between free and A-II complexed forms in very-low- and high-density lipoproteins: functional implications.

The stability of apolipoprotein E/lipoprotein associations has been examined as a function of apolipoprotein E phenotype. Visualisation by immunoblotting showed plasma apolipoprotein E to be present in two forms; the free form and, as previously described, an E-A-II complex. In very low density lipoproteins isolated by gel filtration from subjects with E3/3 and E4/3 phenotypes, apolipoprotein E was present essentially in the free form (ratio free: complex of 12.2 and 37.5, respectively). Exploiting ultracentrifugation as the disruptive agent, very-low-density lipoproteins thus isolated were shown to have substantially lower ratios (5.6 and 5.4, respectively) reflecting preferential loss of free apolipoprotein E. In high-density lipoproteins isolated by gel filtration from E3/3 phenotypes, apolipoprotein E was largely present as an E-A-II complex (80.3%). In contrast, the majority of apolipoprotein E in high-density lipoproteins from E4/3 phenotypes was present in the free form (58.7%). In both phenotypes, the content of free apolipoprotein E was markedly reduced by ultracentrifugation. The results confirm the notion that the formation of the E-A-II complex is a major determinant of the stability of apolipoprotein E-high-density lipoprotein associations. Moreover, that the predominant, ancestral isoform, apolipoprotein E3, exists largely as an E-A-II complex in higher density lipoproteins has important functional implications for this plasma source of apolipoprotein E.

A novel high-density lipoprotein particle and associated protein in rat plasma.

This report describes the characterization of a novel rat apolipoprotein, which, as partial sequencing suggests, does not correspond to any described protein. The protein (termed PX) has an estimated molecular mass of 19.5 kDa and pI in the range 5.5-5.8. Monoclonal antibodies were obtained against protein PX and results on distribution among rat lipoproteins show it to be associated mainly with high-density lipoproteins (HDL), but also with VLDL. Immunoaffinity chromatography of total HDL shows protein PX to be included in a distinct lipoprotein particle, particularly enriched in free cholesterol, with which only traces of other apolipoproteins are associated. Immunologically crossreacting entities are found in the plasma of several species, including man. Retention of the epitope carried by the protein PX would suggest that it is of particular structural or functional importance. It remains to be established whether its function is associated with lipid metabolism.

Actin stress fiber content of regenerated endothelial cells correlates with intramural retention of intermediate plus low density lipoproteins in rat aorta after balloon injury.

The rat aortic model of endothelial injury (balloon catheter induced) has been used to establish whether changes in protein intramural penetration in specific areas of the injured aorta were accompanied by phenotypic modifications of the regenerated endothelial cells covering these particular regions. Iodinated lipoproteins (IDL/LDL fraction) and albumin were used as tracers to localize protein permeability and retention in the aorta. Lipoproteins, but not albumin, were retained in the thickened areas covered with regenerated endothelium (i.e., 60 days after balloon induced injury). Neither lipoproteins nor albumin were retained in the other aortic areas studied, including the intimal thickening of de-endothelialized areas (15 days after injury). The relative volume of cytoplasmic stress fibers was significantly increased in regenerated endothelium covering thickened areas as compared with the other regions of the injured or normal aorta. The accumulation of lipids usually observed in atherosclerotic lesions, compatible with the trapping of lipoproteins by the matrix component of the intimal thickening, may be related to modulated features of endothelial cells regenerated over thickened areas of the aorta.

The membrane-anchoring systems of vertebrate acetylcholinesterase and variant surface glycoproteins of African trypanosomes share a common antigenic determinant.

Amphiphilic detergent-soluble acetylcholinesterase (AChE) from Torpedo is converted to a hydrophilic form by digestion with phospholipase C from Trypanosoma brucei or from Bacillus cereus. This lipase digestion uncovers an immunological determinant which crossreacts with a complex carbohydrate structure present in the hydrophilic form of all variant surface glycoproteins (VSG) of T. brucei. This crossreacting determinant is also detected in human erythrocyte AChE after digestion with T. brucei lipase. From these results we conclude that the glycophospholipid anchors of protozoan VSG and of AChE of the two vertebrates share common structural features, suggesting that this novel type of membrane anchor has been conserved during evolution.