Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP)

Sphingolipid activator proteins SAP-A, -B, -C and -D (also called saposins) are generated by proteolytic processing from a 73 kDa precursor and function as obligatory activators of lysosomal enzymes involved in glycosphingolipid metabolism. Although the SAP precursor can be recognized by the mannose-6-phosphate (M-6-P) receptor and shuttled directly from the secretory pathway to the lysosome, a substantial fraction of newly synthesized precursor is secreted from the cell where it may participate in sphingolipid transport and signaling events. Re-uptake of the secreted precursor is mediated by high-affinity cell surface receptors that are apparently distinct from the M-6-P receptor. We found that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic receptor that is expressed on most cells, can mediate cellular uptake and lysosomal delivery of SAP precursor. Additional in vivo experiments in mice revealed that the mannose receptor system on macrophages also participates in precursor internalization. We conclude that SAP precursor gains entry into cells by at least three independent receptor mechanisms including the M-6-P receptor, the mannose receptor and LRP.     

New evidence for two MHC class II-restricted antigen presentation pathways by overexpression of a small G protein

Exogenous Ags may be presented by MHC class II molecules through two distinct pathways distinguished by their sensitivity to drugs that inhibit the protein synthesis. Using this approach, we previously showed that the subunits Ig-alpha and Ig-beta, associated to B cell Ag receptor, targeted Ags either to newly synthesized or to preexisting pools of MHC class II molecules, respectively. To further characterize these two Ag presentation pathways, we altered the intra-Golgi transport of newly synthesized MHC class II by stably overexpressing, in B cells, mutants of a small G protein involved in the intra-Golgi transport, Rab6. Overexpression of GTP-bound rab6 (Q72L) mutant proteins reduced the cell surface arrival of MHC class II molecules and consequently slowed down Ag presentation dependent upon newly synthesized class II molecules. In contrast, this mutant had no effect on Ag presentation dependent upon preexisting pools of class II molecules, and the overexpression of an inactive GDP-bound form of rab6 (T27N) did not affect any Ag presentation pathway. MHC class II-restricted Ag presentation pathways can therefore be distinguished by their sensitivity to the overexpression of proteins modifying the intracellular transport of newly synthesized class II molecules.     

Functional characterization of the N-glycosylation sites of human acid sphingomyelinase by site-directed mutagenesis

Most soluble lysosomal enzymes require a mannose-6-phosphate recognition marker present on asparagine-linked oligosaccharides for proper targeting to lysosomes. We have determined the influence of the six potential N-linked oligosaccharide chains of human acid sphingomyelinase (ASM) on catalytic activity, targeting, and processing of the enzyme. Each N-glycosylation site was modified by site-directed mutagenesis and subsequently expressed in COS-1 cells. Evidence is presented that five of these sites are used. Elimination of the four N-terminal glycosylation sites does not disturb lysosomal targeting, processing, or enzymatic activity. However, removal of the two C-terminal N-glycosylation sites inhibits the formation of mature enzyme. Absence of glycosylation site five resulted in rapid cleavage of the primary translation product to an enzymatically inactive protein which accumulated inside the endoplasmic reticulum/Golgi, whereas deletion of glycosylation site six led to the formation of an inactive ASM precursor, also retained inside the endoplasmic reticulum/Golgi. Our results also provide evidence that the site of early proteolytic cleavage of newly synthesized ASM must be located between the second and third glycosylation sites.     

Biosynthesis, processing, and targeting of sphingolipid activator protein (SAP )precursor in cultured human fibroblasts. Mannose 6-phosphate receptor-independent endocytosis of SAP precursor

Sphingolipid activator proteins (SAPs) are essential cofactors for the lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acidic exohydrolases. SAP-A, -B, -C, and -D derive from proteolysis of a 73-kDa glycoprotein, the SAP precursor. In the present publication, we studied the intracellular transport and the endocytosis of SAP precursor in human skin fibroblasts. Our data indicate that SAP precursor bears phosphate residues on noncomplex carbohydrate chains linked to the SAP-C and the SAP-D domain and sulfate residues on complex carbohydrate chains located within the SAP-A, -C, and possibly the SAP-D domain. Treatment of fibroblasts with either bafilomycin A1 or 3-methyladenine indicates that proteolytic cleavage of SAP precursor begins as early as in the late endosomes. To determine whether targeting of SAP precursor depends on mannose 6-phosphate residues, we analyzed the processing of SAP precursor in I-cell disease fibroblasts. In these cells nearly normal amounts of newly synthesized SAP-C were found, although secretion of SAP precursor was enhanced 2-3-fold. Moreover, SAP-C could be localized to lysosomal structures by indirect immunofluorescence in normal and in I-cell disease fibroblasts. Mannose 6-phosphate was not found to interfere significantly with endocytosis of SAP precursor. Normal fibroblasts internalized SAP precursor secreted from I-cells nearly as efficiently as the protein secreted from normal cells. To our surprise, deglycosylated SAP precursor was taken up by mannose 6-phosphate receptor double knock out mouse fibroblasts more efficiently than the glycosylated protein. We propose that intracellular targeting of SAP precursor to lysosomes is only partially dependent on mannose 6-phosphate residues, whereas its endocytosis occurs in a carbohydrate-independent manner.     

Phosphorylation regulates the delivery of MHC class II invariant chain complexes to antigen processing compartments

Transport of newly synthesized MHC class II glycoproteins to endosomal Ag processing compartments is mediated by their association with the invariant chain (Ii). Targeting to these compartments is dependent upon recognition of leucine-based endo. somal/lysosomal targeting motifs in the Ii cytosolic domain. Ii, like many molecules that contain leucine-based endosomal targeting motifs, is phosphorylated in vivo. In this report we demonstrate that the cytosolic domain of the p35 Ii isoform is phosphorylated in class II Ii complexes isolated from human B lymphoblastoid cell lines or freshly obtained PBMC. Mutation of serine residue 6 or 8 prevents phosphorylation of Ii-p35 expressed in HeLa cells. Treatment of B lymphoblastoid cell lines with the serine/threonine kinase inhibitor staurosporine prevented Ii phosphorylation and significantly delayed trafficking of newly synthesized class II Ii complexes to endosomal Ag processing compartments. By contrast, staurosporine had no effect on the rate of transport of class I or class II glycoproteins through the Golgi apparatus and did not inhibit the delivery of the chimeric molecule Tac-DM, to endocytic compartments, suggesting that staurosporine does not nonspecifically inhibit protein transport to the endocytic pathway. These results demonstrate that phosphorylation regulates the efficient targeting of MHC class II Ii complexes to Ag processing compartments and strongly suggest that this effect is mediated by phosphorylation of the MHC class II-associated Ii chain.     

Insulin-like growth factor II signaling through the insulin-like growth factor II/mannose-6-phosphate receptor promotes exocytosis in insulin-secreting cells

The insulin-like growth factor II (IGF-II)/mannose-6-phosphate (M-6-P) receptor is known to participate in endocytosis as well as sorting of lysosomal enzymes and is involved in membrane trafficking through rapid cycling between cytosolic membrane compartments and the plasma membrane. Here we demonstrate that IGF-II, acting through the IGF-II/M-6-P receptor, promotes exocytosis of insulin in the pancreatic beta cell. The effect of IGF-II was evoked at nonstimulatory concentrations of glucose, was mediated by a pertussis toxin sensitive GTP-binding protein, was dependent on protein kinase C-induced phosphorylation, and was independent of changes in cytoplasmic free Ca2+ concentration. Since the applied concentration of IGF-II is within the range normally found free in circulation in humans, this novel signaling pathway for the IGF-II/M-6-P receptor is likely to be involved in modulation of insulin exocytosis under physiological conditions.     

Evidence for the requirement of protein synthesis and protein kinase activity in the temperature regulated stability of a Tetrahymena surface protein mRNA

In Tetrahymena thermophila, the expression of the temperature-specific surface protein SerH3 is controlled primarily by a temperature-dependent change in the stability of its mRNA. The change in SerH3 mRNA stability occurs very rapidly after a shift in incubation temperature. This change in temperature could affect SerH3 mRNA stability directly by producing structural changes in the mRNA or regulatory factors acting on SerH3 mRNA. Alternatively, the temperature change could act indirectly through a signal transduction pathway leading to de novo synthesis of new regulatory factors or modifications of existing regulatory factors. To address these issues, we monitored the effect of temperature on an in vitro SerH3 mRNA decay assay and the in vivo effects of a variety of inhibitors against protein synthesis and protein kinases on SerH3 mRNA stability. The results of Northern analysis of SerH3 mRNAs in an in vitro mRNA decay assay indicate that temperature alone can not change the half-life of this mRNA. Furthermore, slot blot analysis of cytoplasmic RNAs show that protein synthesis and the action of protein kinases are not required for SerH3 mRNA turnover in cells grown at 30 degrees C. In contrast, our results indicate that the rapid decay of the SerH3 mRNA in cells grown at 30 degrees C and shifted to 40 degrees C requires a one time serine/threonine phosphorylation event which occurs at the temperature shift. In addition, the data show that a regulatory protein involved in rapid SerH3 mRNA decay must be newly and continuously synthesized following the temperature shift from 30 to 40 degrees C. These data show the complexity of temperature regulated mRNA decay and indicate that phosphorylation and protein synthesis are major factors in this process.     

A dominant-negative clathrin mutant differentially affects trafficking of molecules with distinct sorting motifs in the class II major histocompatibility complex (MHC) pathway

The role of clathrin in intracellular sorting was investigated by expression of a dominant-negative mutant form of clathrin, termed the hub fragment. Hub inhibition of clathrin-mediated membrane transport was established by demonstrating a block of transferrin internalization and an alteration in the intracellular distribution of the cation-independent mannose-6-phosphate receptor. Hubs had no effect on uptake of FITC-dextran, adaptor distribution, organelle integrity in the secretory pathway, or cell surface expression of constitutively secreted molecules. Hub expression blocked lysosomal delivery of chimeric molecules containing either the tyrosine-based sorting signal of H2M or the dileucine-based sorting signal of CD3gamma, confirming a role for clathrin-coated vesicles (CCVs) in recognizing these signals and sorting them to the endocytic pathway. Hub expression was then used to probe the role of CCVs in targeting native molecules bearing these sorting signals in the context of HLA-DM and the invariant chain (I chain) complexed to HLA-DR. The distribution of these molecules was differentially affected. Accumulation of hubs before expression of the DM dimer blocked DM export from the TGN, whereas hubs had no effect on direct targeting of the DR-I chain complex from the TGN to the endocytic pathway. However, concurrent expression of hubs, such that hubs were building to inhibitory concentrations during DM or DR-I chain expression, caused cell surface accumulation of both complexes. These observations suggest that both DM and DR-I chain are directly transported to the endocytic pathway from the TGN, DM in CCVs, and DR-I chain independent of CCVs. Subsequently, both complexes can appear at the cell surface from where they are both internalized by CCVs. Differential packaging in CCVs in the TGN, mediated by tyrosine- and dileucine-based sorting signals, could be a mechanism for functional segregation of DM from DR-I chain until their intended rendezvous in late endocytic compartments.     

Antigen presentation by MHC class II molecules: invariant chain function, protein trafficking, and the molecular basis of diverse determinant capture

Major histocompatibility complex class II molecules are heterodimeric integral membrane proteins whose primary function is the presentation of antigenic peptides derived from proteins entering the endocytic pathway to CD4+ T lymphocytes. To accomplish this physiologic function, class II molecules must assemble in the secretory pathway without undergoing irreversible ligand association at that site, traffic efficiently to the endocytic pathway, and productively interact with protein ligands in these organelles before their ultimate expression on the plasma membrane. Here we review our work describing how invariant chain promoters the assembly and transport process, the complex itinerary of class II-invariant chain complexes through the endocytic pathway, the role of large protein fragments as substrates for class II binding, and the existence of a second pathway for antigen capture by mature class II molecules that complements that involving newly synthesized dimers. We integrate these observations into a coherent model for the operation of a class II-dependent antigen processing and presentation system able to capture diverse antigenic determinants present in proteins of varying structure.     

Cdc2 kinase directly phosphorylates the cis-Golgi matrix protein GM130 and is required for Golgi fragmentation in mitosis

Mitotic fragmentation of the Golgi apparatus can be largely explained by disruption of the interaction between GM130 and the vesicle-docking protein p115. Here we identify a single serine (Ser-25) in GM130 as the key phosphorylated target and Cdc2 as the responsible kinase. MEK1, a component of the MAP kinase signaling pathway recently implicated in mitotic Golgi fragmentation, was not required for GM130 phosphorylation or mitotic fragmentation either in vitro or in vivo. We propose that Cdc2 is directly involved in mitotic Golgi fragmentation and that signaling via MEK1 is not required for this process.     

The vesicle transport protein Vps33p is an ATP-binding protein that localizes to the cytosol in an energy-dependent manner

Molecular mechanisms of vesicle transport between the prevacuolar compartment and the vacuole in yeast or the lysosome in mammalian cells are poorly understood. To learn more about the specificity of this intercompartmental step, we have examined the subcellular localization of a SEC1 homologue, Vps33p, a protein implicated to function in transport between the prevacuolar compartment and the vacuole. Following short pulses, 80-90% of newly synthesized Vps33p cofractionated with a cytosolic enzyme marker after making permeabilized yeast cells. However, during a chase, 20-40% of Vps33p fractionated with permeabilized cell membranes in a time-dependent fashion with a half-time of approximately 40 min. Depletion of cellular ATP increased the association rate to a half-time of approximately 4 min and caused 80-90% of newly synthesized Vps33p to be associated with permeabilized cell membranes. The association of Vps33p with permeabilized cell membranes was reversible after restoring cells with glucose before permeabilization. The N-ethylmaleimide-sensitive fusion protein homologue, Sec18p, a protein with known ATP binding and hydrolysis activity, displayed the same reversible energy-dependent sedimentation characteristics as Vps33p. We determined that the photosensitive analog, 8-azido-[alpha-32P]ATP, could bind directly to Vps33p with low affinity. Interestingly, excess unlabeled ATP could enhance photoaffinity labeling of 8-azido-[alpha-32P]ATP to Vps33p, suggesting cooperative binding, which was not observed with excess GTP. Importantly, we did not detect significant photolabeling after deleting amino acid regions in Vps33p that show similarity to ATP interaction motifs. We visualized these events in living yeast cells after fusing the jellyfish green fluorescent protein (GFP) to the C terminus of full-length Vps33p. In metabolically active cells, the fully functional Vps33p-GFP fusion protein appeared to stain throughout the cytoplasm with one or two very bright fluorescent spots near the vacuole. After depleting cellular ATP, Vps33p-GFP appeared to localize with a punctate morphology, which was also reversible upon restoring cells with glucose. Overall, these data support a model where Vps33p cycles between soluble and particulate forms in an ATP-dependent manner, which may facilitate the specificity of transport vesicle docking or targeting to the yeast lysosome/vacuole.     

Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease whose defective gene has remained elusive. A molecular basis for LINCL was determined with an approach applicable to other lysosomal storage diseases. When the mannose 6-phosphate modification of newly synthesized lysosomal enzymes was used as an affinity marker, a single protein was identified that is absent in LINCL. Sequence comparisons suggest that this protein is a pepstatin-insensitive lysosomal peptidase, and a corresponding enzymatic activity was deficient in LINCL autopsy specimens. Mutations in the gene encoding this protein were identified in LINCL patients but not in normal controls.     

Loss of cytotoxic T lymphocyte function in Chediak-Higashi syndrome arises from a secretory defect that prevents lytic granule exocytosis

CTLs from patients with Chediak-Higashi syndrome (CHS) are unable to destroy target cells recognized via the TCR. To determine the mechanism responsible for the loss of cytotoxicity, CD8+ CTL clones have been derived from a patient with CHS. Individual CTL clones show poor killing that can be increased in longer assays. However, in the presence of cycloheximide, the small amount of killing observed is abolished, indicating killing arises from newly synthesized proteins, rather than from proteins stored in granules. In this study, we show that the CHS CTL clones express normal levels of the lytic proteins granzyme A, granzyme B, and perforin, which are processed properly during biosynthesis and targeted correctly to giant lytic granules. Despite the difference in size, CHS and normal lytic granules are similar, in that both contain the lysosomal enzyme cathepsin D and the lytic protein granzyme A, and lack the mannose-6-phosphate receptor (MPR). However, unlike normal CTL clones, the CHS CTL clones are unable to secrete their giant granules in which the lytic proteins are stored. After cross-linking the TCR, CHS CTL clones fail to secrete granzyme A, as assayed by both enzyme release and confocal microscopy. We suggest that the defect in CHS lies in a protein that is involved in membrane fusion and is essential for the secretion of lysosomal compartments in certain hemopoietic cells.     

Requirement of GM2 ganglioside activator for phospholipase D activation

Sequence analysis of a heat-stable protein necessary for the activation of ADP ribosylation factor-dependent phospholipase D (PLD) reveals that this protein has a structure highly homologous to the previously known GM2 ganglioside activator whose deficiency results in the AB-variant of GM2 gangliosidosis. The heat-stable activator protein indeed has the capacity to enhance enzymatic conversion of GM2 to GM3 ganglioside that is catalyzed by beta-hexosaminidase A. Inversely, GM2 ganglioside activator purified separately from tissues as described earlier [Conzelmann, E. & Sandhoff, K. (1987) Methods Enzymol. 138, 792-815] stimulates ADP ribosylation factor-dependent PLD in a dose-dependent manner. At higher concentrations of ammonium sulfate, the PLD activator protein apparently substitutes for protein kinase C and phosphatidylinositol 4,5-bisphosphate, both of which are known as effective stimulators of the PLD reaction. The mechanism of action of the heat-stable PLD activator protein remains unknown.     

Methodology for the assessment of activities in primary health care]

Polyribosomal complexes beneath postsynaptic sites on dendrites provide a substrate for local translation of particular mRNAs, but the signals that target mRNAs to synapses remain to be defined. Here, we report that high frequency activation of the perforant path projections to the dentate gyrus causes newly synthesized mRNA for the immediate-early gene (IEG) Arc to localize selectively in activated dendritic segments. Newly synthesized Arc protein also accumulates in the portion of the dendrite that had been synaptically activated. The targeting of Arc mRNA was not disrupted by locally inhibiting protein synthesis, indicating that the signals for mRNA localization reside in the mRNA itself. This novel mechanism through which newly synthesized mRNA is precisely targeted to activated synapses is well suited to play a role in the enduring forms of activity-dependent synaptic modification that require protein synthesis.     

The inactivated plasmid inititator protein RepC/RepC* may have a regulatory role

During replication of the plasmid pT181, the initiator protein RepC is modified by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. Here we show that during in vitro replication, RepC* is radioactively labeled, suggesting that the source of the RepC* oligodeoxynucleotide is the newly synthesized pT181 DNA. The RepC/RepC* heterodimer retains its ability to bind the pT181 double-strand origin and, therefore, it may act as a competitive inhibitor of the RepC homodimer during replication.     

Molecular analysis of the acid sphingomyelinase deficiency in a family with an intermediate form of Niemann-Pick disease

A novel point mutation in the lysosomal acid sphingomyelinase gene has been identified in the recently reported Serbian family with a clinically and biochemically atypical intermediate form of Niemann-Pick disease. The mutation was a T1171-->G transversion resulting in substitution of glycine for normal tryptophan at amino acid residue 391. The coding sequence was otherwise normal. All of the five affected individuals were almost certainly homoallelic, and both of the two obligate heterozygotes studied also carried the same mutation. This mutation is therefore likely to be directly associated with the atypical phenotype of these patients. Expression in COS-1 cells suggested a higher residual activity than that in cultured fibroblasts. A recently developed high-affinity rabbit antihuman sphingomyelinase antibody allowed us to study for the first time the biosynthesis, processing, and targeting of a mutant sphingomyelinase by metabolic labeling of cultured fibroblasts. The mutant enzyme protein was normally synthesized, processed, and routed to the lysosome but was apparently unstable and degraded rapidly once it reached the lysosome. Together with the finding of the relatively high residual activity in COS-1 cells, we interpret our observations to mean that instability and rapid breakdown of the mature mutant enzyme protein, due to the mutation rather than direct inactivation of the catalytic activity, is the primary mechanism for the deficiency of sphingomyelinase activity in these patients. A high prevalence of this mutation in the Serbian population is likely, since the family pedigree indicates that members from four reportedly unrelated families must have contributed the same mutation.     

Deletion of the putative effector region of Era, an essential GTP-binding protein in Escherichia coli, causes a dominant-negative phenotype

Gangliosides are highly immunosuppressive molecules but the mechanism(s) by which they act upon cells remains to be fully defined. Several metabolic products of exogenous gangliosides, including ceramide, have recently been suggested as second messengers in programmed cell death (PCD). Therefore, we have probed the role of gangliosides and ceramides in the induction of PCD and in the inhibition of in vitro lymphoproliferation. PCD was caused only by exogenous ceramides with short fatty acyl groups-d18:1-C2:0 (C2-ceramide, where d18:1 is sphingosine and C2:O is an acetyl group) and d18:1-C6:0 (C6-ceramide, where C6:O is a hexanoyl group). None of the gangliosides studied induced PCD, including naturally occurring GM3, synthetic d18:1-C18:0 GM3 (C18-Cer GM3, where C18:0 is a stearoyl group), or even d18:1-C2:0 GM3 (C2-Cer GM3), which itself contains a PCD-causing ceramide. However, these gangliosides were highly immunosuppressive, inhibiting antigen-induced lymphoproliferation at micromolar concentrations. We conclude that exogenous sphingolipids cause inhibition of lymphoproliferation and PCD by two separate and distinct mechanisms of action.