Sec24 proteins and sorting at the endoplasmic reticulum.
COPII proteins are necessary to generate secretory vesicles at the endoplasmic reticulum. In yeast, the Sec24p protein is the only COPII component in which two close orthologues have been identified. By using gene knock-out in yeast, we found that the absence of one of these Sec24 orthologues resulted in a selective secretion defect for a subset of proteins released into the medium. Data base searches revealed the existence of an entire family of Sec24-related proteins in humans, worms, flies, and plants. We identified and cloned two new human cDNAs encoding proteins homologous to yeast Sec24p, in addition to two human cDNAs already present within the data bases. The entire Sec24 family identified to date is characterized by clusters of highly conserved residues within the 2/3 carboxyl-terminal domain of all the proteins and a divergent amino terminus domain. Human (h) Sec24 orthologues co-immunoprecipitate with hSec23Ap and migrate as a complex by size exclusion chromatography. Immunofluorescence microscopy confirmed that these proteins co-localize with hSec23p and hSec13p. Together, our data suggest that in addition to its role in the shaping up of the vesicle, the Sec23-24p complex may be implicated in cargo selection and concentration. (+info)
PU.1 and USF are required for macrophage-specific mannose receptor promoter activity.
In the current study we report the isolation of 854 base pairs of the rat mannose receptor promoter. Analysis of the sequence revealed one Sp1 site, three PU.1 sites, and a potential TATA box (TTTAAA) 33 base pairs 5' of the transcriptional start site. The tissue specificity of the promoter was determined using transient transfections. The promoter was most active in the mature macrophage cell line NR8383 although the promoter also showed activity in the monocytic cell line RAW. No activity was observed in pre-monocytic cell lines or epithelial cell lines. Mutation of the TTTAAA sequence to TTGGAA resulted in a 50% decrease in activity in transient transfection assays suggesting that the promoter contains a functional TATA box. Using electrophoretic mobility shift assays and mutagenesis we established that the transcription factors Sp1, PU.1, and USF bound to the mannose receptor promoter, but only PU.1 and USF contributed to activation. Transient transfections using a dominant negative construct of USF resulted in a 50% decrease in mannose receptor promoter activity, further establishing the role of USF in activating the rat mannose receptor promoter. Comparison of the rat, mouse, and human sequence demonstrated that some binding sites are not conserved. Gel shifts were performed to investigate differences in protein binding between species. USF bound to the rat and human promoter but not to the mouse promoter, suggesting that different mechanisms are involved in regulation of mannose receptor expression in these species. From these results we conclude that, similar to other myeloid promoters, transcription of the rat mannose receptor is regulated by binding of PU.1 and a ubiquitous factor at an adjacent site. However, unlike other myeloid promoters, we have identified USF as the ubiquitous factor, and demonstrated that the promoter contains a functional TATA box. (+info)
Molecular analysis of the ERGIC-53 gene in 35 families with combined factor V-factor VIII deficiency.
Combined factor V-factor VIII deficiency (F5F8D) is a rare, autosomal recessive coagulation disorder in which the levels of both coagulation factors V and VIII are diminished. The F5F8D locus was previously mapped to a 1-cM interval on chromosome 18q21. Mutations in a candidate gene in this region, ERGIC-53, were recently found to be associated with the coagulation defect in nine Jewish families. We performed single-strand conformation and sequence analysis of the ERGIC-53 gene in 35 F5F8D families of different ethnic origins. We identified 13 distinct mutations accounting for 52 of 70 mutant alleles. These were 3 splice site mutations, 6 insertions and deletions resulting in translational frameshifts, 3 nonsense codons, and elimination of the translation initiation codon. These mutations are predicted to result in synthesis of either a truncated protein product or no protein at all. This study revealed that F5F8D shows extensive allelic heterogeneity and all ERGIC-53 mutations resulting in F5F8D are "null." Approximately 26% of the mutations have not been identified, suggesting that lesions in regulatory elements or severe abnormalities within the introns may be responsible for the disease in these individuals. In two such families, ERGIC-53 protein was detectable at normal levels in patients' lymphocytes, raising the further possibility of defects at other genetic loci. (+info)
ERGIC-53 gene structure and mutation analysis in 19 combined factors V and VIII deficiency families.
Combined factors V and VIII deficiency is an autosomal recessive bleeding disorder associated with plasma levels of coagulation factors V and VIII approximately 5% to 30% of normal. The disease gene was recently identified as the endoplasmic reticulum-Golgi intermediate compartment protein ERGIC-53 by positional cloning, with the detection of two founder mutations in 10 Jewish families. To identify mutations in additional families, the structure of the ERGIC-53 gene was determined by genomic polymerase chain reaction (PCR) and sequence analysis of bacterial artificial chromosome clones containing the ERGIC-53 gene. Nineteen additional families were analyzed by direct sequence analysis of the entire coding region and the intron/exon junctions. Seven novel mutations were identified in 10 families, with one additional family found to harbor one of the two previously described mutations. All of the identified mutations would be predicted to result in complete absence of functional ERGIC-53 protein. In 8 of 19 families, no mutation was identified. Genotyping data indicate that at least two of these families are not linked to the ERGIC-53 locus. Taken together, these results suggest that a significant subset of combined factors V and VIII deficiency is due to mutation in one or more additional genes. (+info)
Mapping of structural determinants for the oligomerization of p58, a lectin-like protein of the intermediate compartment and cis-Golgi.
Shortly after synthesis, p58, the rat homologue of the mannose-binding lectin ERGIC-53/MR60, which localizes to pre-Golgi and cis-Golgi compartments, forms dimers and hexamers, after which an equilibrium of both forms is reached. Mature p58, a type I membrane protein, contains four cysteine residues in the lumenal domain which are capable of forming disulphide bonds. The membrane-proximal half of the lumenal domain consists of four predicted alpha-helical domains, one heavily charged and three amphipathic in nature, all candidates for electrostatic or coiled-coil interactions. Using single-stranded mutagenesis, the cysteines were individually changed to alanines and the contribution of each of the alpha-helical domains was probed by internal deletions. The N-terminal cysteine to alanine mutants, C198A and C238A and the double mutant, C198/238A, oligomerized like the wild-type protein. The two membrane-proximal cysteines were found to be necessary for the oligomerization of p58. Mutants lacking one of the membrane proximal cysteines, either C473A or C482A, were unable to form hexamers, while dimers were formed normally. The C473/482A double mutant formed only monomers. Deletion of any of the individual alpha-helical domains had no effect on oligomerization. The dimeric and hexameric forms bound equally well to D-mannose. The dimeric and monomeric mutants displayed a cellular distribution similar to the wild-type protein, indicating that the oligomerization status played a minimal role in maintaining the subcellular distribution of p58. (+info)
A different intracellular distribution of a single reporter protein is determined at steady state by KKXX or KDEL retrieval signals.
To establish the specific contribution to protein topology of KKXX and KDEL retrieval motifs, we have determined by immunogold electron microscopy and cell fractionation the intracellular distribution at steady state of the transmembrane and anchorless versions of human CD8 protein, tagged with KKXX (CD8-E19) and KDEL (CD8-K), respectively, and stably expressed in epithelial rat cells (Martire, G., Mottola, G., Pascale, M. C., Malagolini, N., Turrini, I., Serafini-Cessi, F., Jackson, M. R., and Bonatti, S. (1996) J. Biol. Chem. 271, 3541-3547). The CD8-E19 protein is represented by a single form, initially O-glycosylated: only about half of it is located in the endoplasmic reticulum, whereas more than 30% of the total is present in the intermediate compartment and cis-Golgi complex. In the latter compartments, CD8-E19 colocalizes with beta-coat protein (COP) (COPI component) and shows the higher density of labeling. Conversely, about 90% of the total CD8-KDEL protein is localized in clusters on the endoplasmic reticulum, where significant co-localization with Sec-23p (COPII component) is observed, and unglycosylated and initially O-glycosylated forms apparently constitute a single pool. Altogether, these results suggest that KKXX and KDEL retrieval motifs have different topological effects on theirs own at steady state: the first results in a specific enrichment in the intermediate compartment and cis-Golgi complex, and the latter dictates residency in the endoplasmic reticulum. (+info)
Type 1 and type 2 cytokine regulation of macrophage endocytosis: differential activation by IL-4/IL-13 as opposed to IFN-gamma or IL-10.
Cytokine regulation of endocytic activity in primary human macrophages was studied to define ultrastructural changes and mechanisms of pinocytic regulation associated with cytokines secreted by activated T cells. The effects of IFN-gamma (type 1) and IL-4/IL-13 and IL-10 (type 2) cytokines on fluid phase and mannose receptor-mediated endocytosis were assessed by horseradish peroxidase and colloidal gold-BSA uptake and computer-assisted morphometric analysis. IL-4 and IL-13 enhanced fluid phase pinocytosis and mannose receptor-mediated uptake by activation of phosphatidylinositol 3-kinase. Inhibition of actin assembly showed that both cytokines exerted actin-dependent and -independent effects. Ultrastructurally, IL-4 and IL-13 increased tubular vesicle formation underneath the plasma membrane and at pericentriolar sites, concurrent with decreased particle sorting to lysosomes. By contrast, IL-10 or IFN-gamma decreased both fluid phase pinocytosis and mannose receptor-mediated uptake. IFN-gamma stimulated increased particle sorting to perinuclear lysosomes, while IL-10 decreased this activity. In summary, our data document differential effects on macrophage endocytic functions by type 1 or type 2 cytokines associated with induction and effector pathways in immunity. (+info)
A comparison of the pharmacological properties of carbohydrate remodeled recombinant and placental-derived beta-glucocerebrosidase: implications for clinical efficacy in treatment of Gaucher disease.
The objective of these studies was to characterize the macrophage mannose receptor binding and pharmacological properties of carbohydrate remodeled human placental-derived and recombinant beta-glucocerebrosidase (pGCR and rGCR, respectively). These are similar but not identical molecules that were developed as enzyme replacement therapies for Gaucher disease. Both undergo oligosaccharide remodeling during purification to expose terminal mannose sugar residues. Competitive binding data indicated carbohydrate remodeling improved targeting to mannose receptors over native enzyme by two orders of magnitude. Mannose receptor dissociation constants (Kd) for pGCR and rGCR were each 13 nmol/L. At 37 degrees C, 95% of the total macrophage binding was mannose receptor specific. In vivo, pGCR and rGCR were cleared from circulation by a saturable pathway. The serum half-life (t1/2) was 3 minutes when less than saturable amounts were injected intravenously (IV) into mice. Twenty minutes postdose, beta-glucocerebrosidase activity increased over endogenous levels in all tissues examined. Fifty percent of the injected activity was recovered. Ninety-five percent of recovered activity was in the liver. Parenchymal cells (PC), Kupffer cells (KC), and liver endothelium cells (LEC) were responsible for 75%, 22%, and 3%, respectively, of the hepatocellular uptake of rGCR and for 76%, 11%, and 12%, respectively, of the hepatocellular uptake of pGCR. Both molecules had poor stability in LEC and relatively long terminal half-lives in PC (t1/2 = 2 days) and KC (t1/2 = 3 days). (+info)