A polymorphism of the complement regulatory protein MCP (membrane cofactor protein or gp45-70)

Membrane cofactor protein (MCP or gp45-70) is a recently described regulatory glycoprotein of the complement system which binds iC3 or C3b and is present on human platelets, T cells, B cells, monocytes, and mononuclear-derived cell lines. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, MCP migrates as a doublet with an Mr of the upper band of 63,000 and the lower band of 58,000. The same pattern was found on all cell populations in a given individual and was stable over time. In order to further characterize the two band pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, MCP was isolated by affinity chromatography or immunoprecipitation from 72 healthy unrelated donors. All individuals expressed both bands and, based on the densitometric scanning of gels, three patterns were noted: upper band predominant in 65%, approximately equal distribution of upper and lower bands in 29%, and lower band predominant in 6%. These observed phenotypic frequencies fit with expectations based on Hardy-Weinberg equilibrium for a two-allele system. Family studies also support this model as none of the 26 offspring had a phenotype that deviated from the expected, assuming an autosomal codominant model of inheritance. These results are consistent with a simple two-allele system that controls the expression of the two bands of MCP.     

ReducedS-adenosylmethionine: Protein-lysineN-methyltransferase activity (protein methylase III) in shiverer mutant mouse brain

Mice with the dysmyelinating mutation shiverer were studied by measuring the activity of two protein methylases and myelin marker enzymes in the brain. It was observed thatS-adenosylmethionine: protein-lysineN-methyltransferase (protein methylase III, EC. 2.1.1.43) activity is significantly reduced in phenotypically affected homozygous shiverer (shi/shi) mutant mouse brain compared to the unaffected heterozygous littermate brain. This reduction in enzyme activity is manifested mainly by reduced formation of trimethyllysine during the in vitro methylation of histone. In contrast, myelin marker enzymes such as 2,3-cyclic nucleotide 3-phosphohydrolase and 5-nucleotidase as well asS-adenosyl-methionine: protein-carboxylO-methyltransferase (protein methylase II, EC. 2.1.1.24) activities were not significantly affected in these strains of mice.     

Biosynthesis and glycosylation of the human complement regulatory protein decay-accelerating factor

The biosynthesis and oligosaccharide structure of the human complement regulatory glycoprotein decay-accelerating factor (DAF) were studied in erythrocytes and cell lines. Initial information relative to carbohydrate moieties of DAF was obtained by enzymatic digestions. The 74,000 Mr erythrocyte DAF was lowered 3000 by endoglycosidase F, whereas endoglycosidase H had no effect, indicating one N-linked complex-type unit. Treatment with endo-alpha-N-acetylgalactosaminidase to remove O-linked oligosaccharides resulted in a 48,000 Mr molecule (67% of the Mr shift being due to sialic acid), which decreased to 45,000 Mr after sequential endoglycosidase F treatment. To additionally define the oligosaccharide structure and identify precursors in biosynthetic pathways, DAF was studied in the HL-60 cell line differentiated by vitamin D toward monocytes. Pulse-chase experiments with [35S]methionine revealed a precursor species of 43,000 Mr that underwent an early post-translational modification to a 46,000 Mr intermediate, and subsequently was chased into a mature species of 80,000 Mr that aligned with 125I surface-labeled DAF from these cells. All three forms of DAF were approximately 3000 lower in Mr in the presence of tunicamycin. The two lower Mr DAF species were sensitive to endoglycosidases F and H but not to neuraminidase or endo-alpha-N-acetylgalactosaminidase. In summary, DAF is synthesized as a 43,000 Mr precursor species containing one N-linked high-mannose unit. Before entering the central region of the Golgi, it is converted to a 46,000 Mr species by an as yet unknown post-translational modification. The 46,000 Mr form is converted to the 74,000 Mr (erythrocyte) or 80,000 Mr (leukocyte) membrane form of DAF by the addition of multiple, sialylated O-linked oligosaccharide chains (responsible for the large electrophoretic mobility shift) and conversion of the single N-linked high-mannose unit to a complex-type structure. The cell-specific Mr variation between red and white blood cells arises during this post-translational modification from the 46,000 Mr biosynthetic intermediate to the mature DAF species expressed on the cell surface.     

Palmitylation of an amino-terminal cysteine motif of protein tyrosine kinases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol-anchored proteins.

Cross-linking of glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins on T cells can trigger cell activation. We and others have shown an association between GPI-anchored proteins and the protein tyrosine kinases (PTKs) p56lck and p59fyn, suggesting a pathway for signaling through GPI-anchored proteins. Studies of decay-accelerating factor (DAF) or CD59 in either the C32 cell line or the HeLa cell line transfected with PTK cDNA demonstrated that the GPI-anchored proteins associated noncovalently with p56lck and p59fyn but not with p60src. Nonmyristylated versions of p56lck and p59fyn also failed to associate with the GPI-anchored proteins. Mutational analysis of the PTK demonstrated that the association with the GPI-anchored proteins mapped to the unique amino-terminal domains of the PTK. A chimeric PTK consisting of the 10 amino-terminal residues of p56lck or p59fyn replacing the corresponding amino acids in p60src was sufficient for association with DAF, but the converse constructs containing the first 10 amino acids of p60src plus the remainder of p56lck or p59fyn did not associate with DAF. Mutation of cysteine to serine at positions 3 and 6 in p59fyn or positions 3 and 5 in p56lck abolished the association of these kinases with DAF. Mutation of serine to cysteine at positions 3 and 6 in p60src conferred on p60src the ability to associate with DAF. Direct labeling with [3H]palmitate demonstrated palmitylation of this amino-terminal cysteine motif in p56lck. Thus, palmitylation of the amino-terminal cysteine residue(s) together with myristylation of the amino-terminal glycine residue defines important motifs for the association of PTKs with GPI-anchored proteins.     

The HeLa cell receptor for enterovirus 70 is decay-accelerating factor (CD55).

Enterovirus 70 (EV70) is a recently emerged human pathogen belonging to the family Picornaviridae. The ability of EV70 to infect a wide variety of nonprimate cell lines in vitro is unique among human enteroviruses. The importance of virus receptors as determinants of viral host range and tropism led us to study the host cell receptor for this unusual picornavirus. We produced a monoclonal antibody (MAb), EVR1, which bound to the surface of HeLa cells and protected them against infection by EV70 but not by poliovirus or by coxsackievirus B3. This antibody also inhibited the binding of [35S]EV70 to HeLa cells. MAb EVR1 did not bind to monkey kidney (LLC-MK2) cells, nor did it protect these cells against virus infection. In Western immunoassays and in immunoprecipitations, MAb EVR1 identified a HeLa cell glycoprotein of approximately 75 kDa that is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Decay-accelerating factor (DAF, CD55) is a 70- to 75-kDa GPI-anchored membrane protein that is involved in the regulation of complement and has also been shown to function as a receptor for several enteroviruses. MAb EVR1 bound to Chinese hamster ovary (CHO) cells constitutively expressing human DAF. Anti-DAF MAbs inhibited EV70 binding to HeLa cells and protected them against EV70 infection. Transient expression of human DAF in murine NIH 3T3 cells resulted in binding of labelled EV70 and stably, transformed NIH 3T3 cells expressing DAF were able to support virus replication. These data indicate that the HeLa cell receptor for EV70 is DAF.     

Mapping of epitopes, glycosylation sites, and complement regulatory domains in human decay accelerating factor.

Decay accelerating factor (DAF, CD55) is a glycophospholipid-anchored membrane protein that protects cells from complement-mediated damage by inhibiting the formation and accelerating the decay of C3/C5 convertases. DAF deletion mutants lacking each of the four short consensus repeats (SCR) or the serine/threonine-rich region (S/T) were created by site-directed mutagenesis. These deletion mutants were expressed by stable transfection in Chinese hamster ovary cells for the purpose of mapping important structural and functional sites in DAF. The epitopes on DAF for 16 murine mAb were mapped by immunoprecipitation studies as follows: SCR1, 6; SCR2, 3; SCR3, 3; SCR4, 3; S/T, 1. Testing of 13 mAb showed complete blocking of DAF function only by 1C6 and 1H4, both directed at SCR3. The single N-linked glycosylation site was confirmed at a location between SCR1 and SCR2, and the multiple O-linked oligosaccharides were localized to the S/T region. Functional activity of DAF mutants was assessed by the ability of these transfected constructs to protect Chinese hamster ovary cells from cytotoxicity induced by rabbit antibody plus human complement. Removal of SCR1 had no effect on DAF function, but individual deletion of SCR2, SCR3, or SCR4 totally abolished DAF function. Surprisingly, deletion of the S/T region totally abrogated DAF function, but this could be restored by a fusion construct placing the four SCR domains of DAF onto the HLA-B44 molecule, implying that the O-glycosylated S/T region serves as an important but nonspecific spacer projecting the DAF functional domains above the plasma membrane. Overall, the creation of DAF deletion mutants has elucidated important structure-function relations in the DAF molecule.     

Influence of glycosylation on allelic and cell-specific Mr variation, receptor processing, and ligand binding of the human complement C3b/C4b receptor

The human complement C3b/C4b receptor (CR1), a single chain membrane glycoprotein of Mr approximately 200,000, has several cell-specific Mr variations as well as an allelic variation involving four phenotypes whose Mr values span a range of 90,000. We investigated the role of glycosylation in these structural variations and in receptor metabolism. In the human HL-60 promyelocytic leukemic cell line differentiated toward granulocytes or monocytes and in Epstein-Barr virus-transformed B lymphoblastoid cell lines of the common phenotype, CR1 is synthesized as a precursor of Mr = 222,000 that is converted into mature CR1s with differing Mr values. Endoglycosidase F treatment of mature CR1 from all these cell types produced the same lower Mr band. Additionally, the previously noted 5,000 higher Mr of CR1 from human peripheral granulocytes versus erythrocytes was abrogated by endoglycosidase F. Hence these cell-specific variations are due to differences in N-glycosylation. Lectin affinity chromatography shows that these N-linked oligosaccharides are mostly tri- and tetraantennary complex-type species with specific differences in fractionation patterns that correlate with their differing Mrs. In lymphoblastoid cell lines, the four allelic variants each have a precursor 6,000 lower in Mr than the respective mature CR1. In the presence of tunicamycin, each of the CR1 allelic products is 25,000 lower in Mr than the glycosylated receptor. The failure to radiolabel CR1 with [3H]glucosamine in the presence of tunicamycin indicates the lack of O-linked oligosaccharide on CR1. These data, taken together, strongly suggest that the CR1 polymorphism resides at the polypeptide level. Nonglycosylated CR1 synthesized in the presence of tunicamycin had twice the turnover rate of glycosylated CR1. The efficiency of surface membrane insertion and of ligand binding to hemolytically inactive C3 were markedly reduced for nonglycosylated CR1, suggesting that glycosylation is important for the proper expression of CR1 function.     

Endocytosis of oxidized low density lipoprotein through scavenger receptor CD36 utilizes a lipid raft pathway that does not require caveolin-1

The scavenger receptor CD36 binds a diverse array of ligands, including thrombospondin-1, oxidized low density lipoprotein (OxLDL), fatty acids, anionic phospholipids, and apoptotic cells. CD36 has been reported to be present in lipid rafts/caveolae, but little is known about the membrane trafficking of this protein at baseline or following ligand binding. Here, we determined that expression of CD36 in Chinese hamster ovary (CHO) cells and endogenous expression of CD36 in C32 cells led to a homogeneous distribution of the protein on the plasma membrane, as judged by confocal fluorescence microscopy. This homogeneous pattern was observed both by anti-CD36 antibody staining and by live cell imaging of CHO cells expressing a chimeric CD36-green fluorescent protein construct. In contrast, caveolin-1 displayed its usual punctate surface distribution. Correspondingly, dual labeling of CD36 and caveolin-1 showed essentially no overlap, neither by immunofluorescence light microscopy nor by immunogold electron microscopy. Furthermore, isolation of lipid rafts by sucrose gradient ultracentrifugation of cold Triton X-100 cell lysates yielded both CD36 and caveolin-1, but immunoprecipitates of caveolin-1 did not contain CD36. Binding of Ox-LDL led to internalization of CD36 and OxLDL into endosomal structures that did not contain caveolin-1 or transferrin but that co-internalized the glycosyl-phosphatidylinositol-anchored protein decay accelerating factor, a lipid raft protein. Furthermore, expression of CD36 in the caveolin-1-negative KB cell line is sufficient for OxLDL-induced internalization of CD36, indicating that caveolin-1 is not required for this endocytic process. Taken together, these data demonstrate that at steady state, CD36 is localized in lipid rafts but not in caveolae, and that binding of OxLDL to CD36 leads to endocytosis through a lipid raft pathway that is distinct from the clathrin-mediated or caveolin internalization pathways.     

Prostanoid receptors in intestinal epithelium: selective expression, function, and change with inflammation

The tissue concentration of PGE(2)is heightened during mucosal inflammation. Nevertheless, the cellular targets of this prostanoid and its effects on epithelial cell physiology are incompletely understood. We used a panel of specific immunoglobulin and mRNA probes in order to localize and quantitate the four member EP family of prostanoid receptors for binding PGE(2)on cells of histologically normal and inflamed human colonic mucosa, and then examined the physiological consequences for the epithelial component of intestine, with special attention to its barrier function. Prostanoid receptors were selectively expressed on a limited number of human colonic mucosal cells, and differed markedly between normal and inflamed tissue. In non-inflamed mucosa, EP(2)and EP(3)were expressed on epithelia at the apex of crypts; while EP(4)was expressed on surface and lateral crypt epithelia. Dual immunostaining and in situ hybridization with digoxygenin-labelled RNA probes largely confirmed the epithelial localization of EP(4). On the other hand, during inflammation, lateral crypt (non-surface) epithelial cells newly and significantly expressed prostanoid receptors EP(2)and EP(3)(p<0.05, by computer-assisted densitometry). Functionally, exogenous E series prostanoids applied to epithelial monolayers in nM concentrations brought about a 24% increase in the level of barrier function; an associated rise in intracellular cAMP (EC(50)of 281); and protection of epithelium from the effects of T cell cytokines. A major perturbation in the number and distribution of functional eicosonoid receptors on epithelia occurs in chronic inflammation of human colonic mucosa.