Tandem mass tag-based proteomic analysis of endoplasmic reticulum proteins in mulberry leaves under ultraviolet-B and dark stress.

Mulberry leaves have been used in traditional Chinese medicine due to their antioxidant, antidiabetic, and antihyperlipidemic properties. A previous study showed that ultraviolet-B radiation followed by dark incubation could improve the contents of active ingredients in mulberry leaves, such as moracin N and chalcomoracin. The endoplasmic reticulum (ER) serves as a protein quality control center and the location for protein synthesis, which is involved in the response to the environmental stress in plants. To investigate the mechanisms in response to ultraviolet-B radiation followed by dark incubation (UV + D), ER proteomics was performed on mulberry leaves. The ER protein markers, glucose-regulated protein (GRP78), and calnexin (CNX), were significantly higher in the ER fraction than in the total protein fraction, indicating that the ER was purified. Compared to the control, the abundance of protein disulfide isomerase, UDP-glucose glycoprotein glucosyltransferase, CNX, and calreticulin proteins decreased, while of the abundance of heat shock-related proteins increased under stress. P450 enzyme system-related proteins and ribosomal proteins showed significant increases. These results suggest that under UV + D stress, mulberry leaves activated the cell redox and ER quality control systems, enhancing protein synthesis and weakening N-glycan biosynthesis in the ER to resist the damage.

Glycan structure-based perspectives on the entry and release of glycoproteins in the calnexin/calreticulin cycle.

N-glycans are attached to newly synthesised polypeptides and are involved in the folding, secretion, and degradation of N-linked glycoproteins. In particular, the calnexin/calreticulin cycle, which is the central mechanism of the entry and release of N-linked glycoproteins depending on the folding sates, has been well studied. In addition to biological studies on the calnexin/calreticulin cycle, several studies have revealed complementary roles of in vitro chemistry-based research in the structure-based understanding of the cycle. In this mini-review, we summarise chemistry-based results and highlight their importance for further understanding of the cycle.

Systematic deletion of the ER lectin chaperone genes reveals their roles in vegetative growth and male gametophyte development in Arabidopsis.

Calnexin (CNX) and calreticulin (CRT) are homologous lectin chaperones in the endoplasmic reticulum (ER) that facilitate glycoprotein folding and retain folding intermediates to prevent their transit via the secretary pathway. The Arabidopsis genome has two CNX (CNX1 and CNX2) and three CRT (CRT1, CRT2 and CRT3) homologs. Despite growing evidence of the biological roles of CNXs and CRTs, little is understood about their function in Arabidopsis growth and development under normal conditions. Here, we report that the deletion of CNX1, but not of CNX2, in the crt1 crt2 crt3 triple mutation background had an adverse effect on pollen viability and pollen tube growth, leading to a significant reduction in fertility. The cnx1 crt1 crt2 crt3 quadruple mutation also conferred severe defects in growth and development, including a shortened primary root, increased root hair length and density, and reduced plant height. Disruption of all five members of the CNX/CRT family was revealed to be lethal. Finally, the abnormal phenotype of the cnx1 crt1 crt2 crt3 quadruple mutants was completely rescued by either the CNX1 or CNX2 cDNA under the control of the CNX1 promoter, suggesting functional redundancy between CNX1 and CNX2. Taken together, these results provide genetic evidence that CNX and CRT play essential and overlapping roles during vegetative growth and male gametophyte development in Arabidopsis.

Two endoplasmic reticulum proteins (calnexin and calreticulin) are involved in innate immunity in Chinese mitten crab (Eriocheir sinensis).

Calnexin (Cnx) and calreticulin (Crt), which are important chaperones in the endoplasmic reticulum (ER), participate in the folding and quality control of client proteins. Cnx and Crt identified from Chinese mitten crab (Eriocheir sinensis) are designated as EsCnx and EsCrt, respectively. EsCnx and EsCrt are expressed in the hemocyte, hepatopancrea, gill, and intestine at the mRNA and protein level. Immunofluorescence analysis indicated that EsCnx and EsCRT are located in the ER. Moreover, the mRNA and protein expression levels of EsCnx and EsCrt were altered by challenge with lipopolysaccharides (LPS), peptidoglycans (PGN), Staphyloccocus aureus, and Vibrio parahaemolyticus. Recombinant EsCnx and EsCrt (rEsCnx and rEsCrt, respectively) proteins can bind to various Gram-positive and Gram-negative bacteria, as well as to different polysaccharides (LPS and PGN). rEsCnx and rEsCrt assisted in the clearance of V. parahaemolyticus in vivo, and the clearance efficiency was impaired after silencing of EsCnx and EsCrt. Our results suggest that the two ER proteins are involved in anti-bacterial immunity in E. sinensis.

Hepatic Macrosteatosis Is Partially Converted to Microsteatosis by Melatonin Supplementation in ob/ob Mice Non-Alcoholic Fatty Liver Disease.

BACKGROUND: Obesity is a common risk factor for non-alcoholic fatty liver disease (NAFLD). Currently, there are no specific treatments against NAFLD. Thus, examining any molecule with potential benefits against this condition emerged melatonin as a molecule that influences metabolic dysfunctions. The aim of this study was to determine whether melatonin would function against NAFDL, studying morphological, ultrastuctural and metabolic markers that characterize the liver of ob/ob mice. METHODS: Lean and ob/ob mice were supplemented with melatonin in the drinking water for 8 weeks. Histology and stereology were performed to assess hepatic steatosis and glycogen deposition. Ultrastructural features of mitochondria, endoplasmic reticulum (ER) and their juxtapositions were evaluated in livers of all experimental groups. Furthermore, hepatic distribution and expression of markers of ER and mitochondria (calnexin, ATP sintase ß, GRP78 and CHOP) and metabolic dysfunction (RPB4, ß-catenin) and cellular longevity (SIRT1) were analyzed. RESULTS: Melatonin significantly reduced glycemia, identified also by a decrease of hepatic RBP4 expression, reversed macrosteatosis in microsteatosis at the hepatic pericentral zone, enlarged ER-mitochondrial distance and ameliorated the morphology and organization of these organelles in ob/ob mouse liver. Furthermore, in ob/ob mice, calnexin and ATP synthase ß were partially restored, GRP78 and CHOP decreased in periportal and midzonal hepatocytes and ß-catenin expression was, in part, restored in peripheral membranes of hepatocytes. Melatonin supplementation to ob/ob mice improves hepatic morphological, ultrastructural and metabolic damage that occurs as a result of NAFLD. CONCLUSIONS: Melatonin may be a potential adjuvant treatment to limit NAFLD and its progression into irreversible complications.

Global mass spectrometry and transcriptomics array based drug profiling provides novel insight into glucosamine induced endoplasmic reticulum stress.

We investigated the molecular effects of glucosamine supplements, a popular and safe alternative to nonsteroidal anti-inflammatory drugs, for decreasing pain, inflammation, and maintaining healthy joints. Numerous studies have reported an array of molecular effects after glucosamine treatment. We questioned whether the differences in the effects observed in previous studies were associated with the focus on a specific subproteome or with the use of specific cell lines or tissues. To address this question, global mass spectrometry- and transcription array-based glucosamine drug profiling was performed on malignant cell lines from different stages of lymphocyte development. We combined global label-free MS-based protein quantitation with an open search for modifications to obtain the best possible proteome coverage. Our data were largely consistent with previous studies in a variety of cellular models. We mainly observed glucosamine induced O-GlcNAcylation/O-GalNAcylation (O-HexNAcylation); however, we also observed global and local changes in acetylation, methylation, and phosphorylation. For example, our data provides two additional examples of "yin-yang" between phosphorylation and O-HexNAcylation. Furthermore, we mapped novel O-HexNAc sites on GLU2B and calnexin. GLU2B and calnexin are known to be located in the endoplasmic reticulum (ER) and involved in protein folding and quality control. The O-HexNAc sites were regulated by glucosamine treatment and correlated with the up-regulation of the ER stress marker GRP78. The occupancy of O-HexNAc on GLU2B and calnexin sites differed between the cytosolic and nuclear fractions with a higher occupancy in the cytosolic fraction. Based on our data we propose the hypothesis that O-HexNAc either inactivates calnexin and/or targets it to the cytosolic fraction. Further, we hypothesize that O-HexNAcylation induced by glucosamine treatment enhances protein trafficking.

Selenoprotein S is a marker but not a regulator of endoplasmic reticulum stress in intestinal epithelial cells.

Selenoproteins are candidate mediators of selenium-dependent protection against tumorigenesis and inflammation in the gut. Expression and roles of only a limited number of intestinal selenoproteins have been described so far. Selenoprotein S (SelS) has been linked to various inflammatory diseases and is suggested to be involved in endoplasmic reticulum (ER) homeostasis regulation and antioxidative protection in a cell-type-dependent manner, but its protein expression, regulation, and function in the gut are not known. We here analyzed the expression and localization of SelS in the healthy and inflamed gut and studied its regulation and function in intestinal epithelial cell lines. SelS was expressed in the intestinal epithelium of the small and large intestine and colocalized with markers of Paneth cells and macrophages. It was upregulated in inflamed ileal tissue from Crohn's disease patients and in two models of experimental colitis in mice. We detected SelS in colorectal cell lines, where it colocalized with the ER marker calnexin. SelS protein expression was unaffected by enterocytic differentiation but increased in response to selenium supplementation and after treatment with the ER stress inducer tunicamycin. On the other hand, depletion of SelS in LS174T, HT29, and Caco-2 cells by RNA interference did not cause or modulate ER stress and had no effect on hydrogen peroxide-induced cell death. In summary, we introduce SelS as a novel marker of Paneth cells and intestinal ER stress. Although it is upregulated in Crohn's disease, its role in disease etiology remains to be established.

Calcineurin interacts with PERK and dephosphorylates calnexin to relieve ER stress in mammals and frogs.

BACKGROUND: The accumulation of misfolded proteins within the endoplasmic reticulum (ER) triggers a cellular process known as the Unfolded Protein Response (UPR). One of the earliest responses is the attenuation of protein translation. Little is known about the role that Ca2+ mobilization plays in the early UPR. Work from our group has shown that cytosolic phosphorylation of calnexin (CLNX) controls Ca2+ uptake into the ER via the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) 2b. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that calcineurin (CN), a Ca2+ dependent phosphatase, associates with the (PKR)-like ER kinase (PERK), and promotes PERK auto-phosphorylation. This association, in turn, increases the phosphorylation level of eukaryotic initiation factor-2 alpha (eIF2-alpha) and attenuates protein translation. Data supporting these conclusions were obtained from co-immunoprecipitations, pull-down assays, in-vitro kinase assays, siRNA treatments and [35S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca2+ concentrations and involved the cytosolic domain of PERK. CN levels were rapidly increased by ER stressors, which could be blocked by siRNA treatments for CN-Aalpha in cultured astrocytes. Downregulation of CN blocked subsequent ER-stress-induced increases in phosphorylated elF2-alpha. CN knockdown in Xenopus oocytes predisposed them to induction of apoptosis. We also found that CLNX was dephosphorylated by CN when Ca2+ increased. These data were obtained from [gamma32P]-CLNX immunoprecipitations and Ca2+ imaging measurements. CLNX was dephosphorylated when Xenopus oocytes were treated with ER stressors. Dephosphorylation was pharmacologically blocked by treatment with CN inhibitors. Finally, evidence is presented that PERK phosphorylates CN-A at low resting levels of Ca2+. We further show that phosphorylated CN-A exhibits decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is re-established. CONCLUSIONS/SIGNIFICANCE: Our data suggest two new complementary roles for CN in the regulation of the early UPR. First, CN binding to PERK enhances inhibition of protein translation to allow the cell time to recover. The induction of the early UPR, as indicated by increased P-elF2alpha, is critically dependent on a translational increase in CN-Aalpha. Second, CN dephosphorylates CLNX and likely removes inhibition of SERCA2b activity, which would aid the rapid restoration of ER Ca2+ homeostasis.

Molecular cloning of calnexin and calreticulin in the Pacific oyster Crassostrea gigas and its expression in response to air exposure.

Calnexin (CNX) and calreticulin (CRT) are endoplasmic reticulum (ER) chaperones. CNX is a type I transmembrane protein and CRT is a soluble CNX homologue. In the ER, CNX and CRT are important for Ca(2+) homeostasis and protein maturation. Here, we describe the full-length cDNA of the first mollusk CNX (cgCNX) and a second mollusk CRT (cgCRT) from the oyster Crassostrea gigas. CgCNX, containing 3255bp, was composed of a 1764bp open reading frame (ORF) that encodes a 588-amino acid protein. CgCRT, containing 1727bp, was composed of a 1242bp ORF that encodes a 414-amino acid protein. CgCNX and cgCRT contains an N-terminal 21- and 16-amino acid sequence, respectively, which is characteristic of a signal sequence. At the C-terminus, cgCRT also contains the KDEL (-Lys-Asp-Glu-Leu) peptide motif suggesting that cgCRT localizes in the ER. Northern blot analysis showed that both cgCNX and cgCRT mRNAs are induced by air exposure. The expression patterns of cgCNX mRNA differed from those of cgCRT during air exposure. This suggests that these two molecular chaperones have different roles in the response to air exposure.

A novel calcium oxalate crystal growth inhibitory protein from the seeds of Dolichos biflorus (L.).

Recurrence and persistent side effects of present day treatment for urolithiasis restrict their use, so an alternate, using phytotherapy is being sought. Dolichos biflorus seeds, which are used as dietary food in India, possess antilithiatic properties. In the present study, a novel dimeric antilithiatic protein (98 kDa) from its seeds was purified based on its ability to inhibit calcium oxalate crystallization in vitro. Amino acid analysis of Dolichos biflorus antilithiatic protein showed abundant acidic amino acids. The mascot search engine presented sequence similarity with a calcium binding protein, calnexin of Pisum sativum from the m/z data obtained by MALDI TOF mass spectrometer. Above results demonstrate the anticalcifying/antilithiatic nature of a novel protein from the seeds of Dolichos biflorus and thus open new vistas for using plant proteins as therapeutic agents to treat urolithiasis.

Alpha-tocopherol induces calnexin in renal tubular cells: another protective mechanism against free radical-induced cellular damage.

Pre-administration of alpha-tocopherol is protective against oxidative renal tubular damage and subsequent carcinogenesis by ferric nitrilotriacetate (Fe-NTA) in rats. We searched for mechanisms other than the scavenging effect of alpha-tocopherol with microarray analyses, which implicated calnexin, a chaperone for glycoproteins. Renal mRNA levels of calnexin significantly increased 3h after an injection of Fe-NTA in rats fed a standard diet whereas those fed an alpha-tocopherol-supplemented diet showed an increase prior to injection, but after injection showed a decrease in renal calnexin mRNA levels, with unaltered protein levels. In experiments using LLC-PK1 cells, addition of alpha-tocopherol was protective against oxidative stress by H2O2, concomitant with calnexin induction. Knockdown of calnexin by siRNA significantly reduced this protection. Furthermore, COS-7 cells transfected with the calnexin gene were more resistant to H2O2. Together with the fact that alpha-tocopherol induced N-acetylglucosaminyltransferase 3, our data suggest that alpha-tocopherol modifies glycoprotein metabolism partially by conferring mild ER stress. This adds another molecular mechanism of alpha-tocopherol toward cancer prevention.

Characterization of endoplasmic reticulum-associated degradation of a protein S mutant identified in a family of quantitative protein S deficiency.

INTRODUCTION: Misfolded and unassembled glycoproteins are eliminated from the endoplasmic reticulum (ER) lumen by the ER-associated degradation (ERAD). We previously identified a Tyr595Cys (Y595C) mutation of protein S (PS) in a family of a quantitative PS deficiency. The mutation causes intracellular degradation and decreased secretion of the Y595C mutant PS. The aim of the present study was to further characterize the molecular basis of the intracellular degradation of the mutant. MATERIALS AND METHODS: We stably expressed the mutant in mammalian cells, and analyzed the intracellular localization of the protein. The intracellular degradation pathway was determined by pulse-chase analyses in the presence of various inhibitors of ERAD. RESULTS AND CONCLUSIONS: Endoglycosidase H digestion and immunofluorescence staining revealed the mutant being retained in the ER. Epoxomicin, a potent and specific proteasome inhibitor, and Ala-Ala-Phe-CH(2)Cl (AAF), an inhibitor of tripeptidyl peptidase II (TPPII), suppressed the intracellular degradation of the mutant by about 65% and 50%, respectively. When epoxomicin was combined with AAF, the inhibitory effect was substantially enhanced. Although castanospermine, an inhibitor of glucosidases I and II, did not affect the degradation, kifunensine, an inhibitor of ER mannosidase I, suppressed it. Thus, it appears that the Y595C mutant is degraded through more than one pathway of ERAD, including the proteasome-dependent pathway and an alternate proteasome-independent pathway where proteases such as TPPII may be involved. Production of the critical B isoform of Man(8)GlcNAc(2) targets the mutant for ERAD, however, the interaction with calnexin/calreticulin through monoglucosylated oligosaccharides may not be required for the degradation of the mutant.

Mixed-disulfide folding intermediates between thyroglobulin and endoplasmic reticulum resident oxidoreductases ERp57 and protein disulfide isomerase.

We present the first identification of transient folding intermediates of endogenous thyroglobulin (Tg; a large homodimeric secretory glycoprotein of thyrocytes), which include mixed disulfides with endogenous oxidoreductases servicing Tg folding needs. Formation of disulfide-linked Tg adducts with endoplasmic reticulum (ER) oxidoreductases begins cotranslationally. Inhibition of ER glucosidase activity blocked formation of a subgroup of Tg adducts containing ERp57 while causing increased Tg adduct formation with protein disulfide isomerase (PDI), delayed adduct resolution, perturbed oxidative folding of Tg monomers, impaired Tg dimerization, increased Tg association with BiP/GRP78 and GRP94, activation of the unfolded protein response, increased ER-associated degradation of a subpopulation of Tg, partial Tg escape from ER quality control with increased secretion of free monomers, and decreased overall Tg secretion. These data point towards mixed disulfides with the ERp57 oxidoreductase in conjunction with calreticulin/calnexin chaperones acting as normal early Tg folding intermediates that can be "substituted" by PDI adducts only at the expense of lower folding efficiency with resultant ER stress.

Enamel matrix protein interactions.

UNLABELLED: The recognized structural proteins of the enamel matrix are amelogenin, ameloblastin, and enamelin. While a large volume of data exists showing that amelogenin self-assembles into multimeric units referred to as nanospheres, other reports of enamel matrix protein-protein interactions are scant. We believe that each of these enamel matrix proteins must interact with other organic components of ameloblasts and the enamel matrix. Likely protein partners would include integral membrane proteins and additional secreted proteins. INTRODUCTION: The purpose of this study was to identify and catalog additional proteins that play a significant role in enamel formation. MATERIALS AND METHODS: We used the yeast two-hybrid assay to identify protein partners for amelogenin, ameloblastin, and enamelin. Once identified, RT-PCR was used to assess gene transcription of these newly identified and potential "enamel" proteins in ameloblast-like LS8 cells. RESULTS: In the context of this yeast assay, we identified a number of secreted proteins and integral membrane proteins that interact with amelogenin, ameloblastin, and enamelin. Additionally, proteins whose functions range from the inhibition of soft tissue mineralization, calcium ion transport, and phosphorylation events have been identified as protein partners to these enamel matrix proteins. For each protein identified using this screening strategy, future studies are planned to confirm this physiological relationship to biomineralization in vivo. CONCLUSION: Identifying integral membrane proteins of the secretory surface of ameloblast cells (Tomes' processes) and additional enamel matrix proteins, based on their abilities to interact with the most abundant enamel matrix proteins, will better define the molecular mechanisms of enamel formation at its most rudimentary level.

A choline-deficient diet in mice inhibits neither the CDP-choline pathway for phosphatidylcholine synthesis in hepatocytes nor apolipoprotein B secretion.

Phosphatidylcholine is a major component of very low density lipoproteins (VLDLs) secreted by the liver. Hepatic phosphatidylcholine is synthesized from choline via the CDP-choline pathway and from the phosphatidylethanolamine N-methyltransferase pathway. Elimination of the methyltransferase in male mice reduces hepatic VLDL secretion. Our objective was to determine whether inhibition of the CDP-choline pathway for phosphatidylcholine synthesis (by restricting the supply of choline) also impaired VLDL secretion. In mice fed a choline-deficient (CD), compared with a choline-supplemented, diet for 21 days, the amounts of plasma apolipoproteins (apo) B100 and B48 were reduced and the liver triacylglycerol content was increased. Hepatocytes were isolated from male mice that had been fed the CD diet for 3 or 21 days, and the cells were incubated with or without choline. The secretion of apoB100 and B48 from CD hepatocytes was not reduced, and triacylglycerol secretion was only modestly decreased, compared with that from cells supplemented with choline. Remarkably, in light of widely held assumptions, the rate of phosphatidylcholine synthesis from the CDP-choline pathway was not decreased in CD hepatocytes. Rather, there was a trend toward increased phosphatidylcholine synthesis that might be explained by enhanced CTP:phosphocholine cytidylyltransferase activity. Although the concentration of phosphocholine in CD hepatocytes was reduced, the size of the phosphocholine pool remained well above the K for the cytidylyltransferase. Moreover, the amount and m activity of the cytidylyltransferase and methyltransferase were increased. The reduction in plasma apoB in mice deprived of dietary choline cannot, therefore, be attributed to decreased apoB secretion.

Molecular biology of deregulated gene expression in transformed C3H/10T1/2 mouse embryo cell lines induced by specific insoluble carcinogenic nickel compounds.

In the past, exposure of workers to mixtures of soluble and insoluble nickel compounds by inhalation during nickel refining correlated with increased incidences of lung and nasal cancers. Insoluble nickel subsulfide and nickel oxide (NiO) are carcinogenic in animals by inhalation; soluble nickel sulfate is not. Particles of insoluble nickel compounds were phagocytized by C3H/10T1/2 mouse embryo cells and induced morphological transformation in these cells with the following order of potency: NiO (black) > NiO (green) > nickel subsulfide. Foci induced by black/green NiO and nickel monosulfide developed into anchorage-independent transformed cell lines. Random arbitrarily primed-polymerase chain reaction mRNA differential display showed that nine c-DNA fragments are differentially expressed between nontransformed and nickel compound-transformed 10T1/2 cell lines in 6% of total mRNA; 130 genes would be differentially expressed in 100% of the mRNA. Fragment R3-2 was a sequence in the mouse calnexin gene, fragment R3-1 a portion of the Wdr1 gene, and fragment R2-4 a portion of the ect-2 protooncogene. These three genes were overexpressed in transformed cell lines. Fragment R1-2 was 90% homologous to a fragment of the DRIP/TRAP-80 (vitamin D receptor interacting protein/thyroid hormone receptor-activating protein 80) genes and was expressed in nontransformed but not in nickel-transformed cell lines. Specific insoluble carcinogenic nickel compounds are phagocytized into 10T1/2 cells and likely generate oxygen radicals, which would cause mutations in protooncogenes, and chromosome breakage, and mutations in tumor suppressor genes, inactivating them. These compounds also induce methylation of promoters of tumor suppressor genes, inactivating them. This could lead to permanent overexpresssion of the ect-2, calnexin, and Wdr1 genes and suppression of expression of the DRIP/TRAP-80 gene that we observed, which likely contribute to induction and maintenance of transformed phenotypes.

Calnexin overexpression increases manganese peroxidase production in Aspergillus niger.

Heme-containing peroxidases from white rot basidiomycetes, in contrast to most proteins of fungal origin, are poorly produced in industrial filamentous fungal strains. Factors limiting peroxidase production are believed to operate at the posttranslational level. In particular, insufficient availability of the prosthetic group which is required for peroxidase biosynthesis has been proposed to be an important bottleneck. In this work, we analyzed the role of two components of the secretion pathway, the chaperones calnexin and binding protein (BiP), in the production of a fungal peroxidase. Expression of the Phanerochaete chrysosporium manganese peroxidase (MnP) in Aspergillus niger resulted in an increase in the expression level of the clxA and bipA genes. In a heme-supplemented medium, where MnP was shown to be overproduced to higher levels, induction of clxA and bipA was also higher. Overexpression of these two chaperones in an MnP-producing strain was analyzed for its effect on MnP production. Whereas bipA overexpression seriously reduced MnP production, overexpression of calnexin resulted in a four- to fivefold increase in the extracellular MnP levels. However, when additional heme was provided in the culture medium, calnexin overexpression had no synergistic effect on MnP production. The possible function of these two chaperones in MnP maturation and production is discussed.

Maturation of lipoprotein lipase in the endoplasmic reticulum. Concurrent formation of functional dimers and inactive aggregates.

The maturation of lipoprotein lipase (LPL) into a catalytically active enzyme was believed to occur only after its transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To test this hypothesis, LPL located in these two subcellular compartments was separated and compared. Heparin affinity chromatography resolved low affinity, inactive LPL displaying ER characteristics from a high affinity, active fraction exhibiting both ER and Golgi forms. The latter forms were further separated by beta-ricin chromatography and were found to have comparable activities per unit of LPL mass. Thus, LPL must reach a functional conformation in the ER. Active LPL, regardless of its cellular location, exhibited the expected dimer conformation. However, inactive LPL, found only in the ER, was highly aggregated. Kinetic analysis indicated a concurrent formation of LPL dimer and aggregate and indicated that the two forms have dissimilar fates. Whereas the dimer remained stable even when confined to the ER, the aggregate was degraded. Degradation rates were not affected by proteasomal or lysosomal inhibitors but were markedly reduced by ATP depletion. Lowering the redox potential in the ER by dithiothreitol caused the dimer to associate with calnexin, BiP, and protein-disulfide isomerase to form large, inactive complexes; dithiothreitol removal induced complex dissociation with restoration of the functional LPL dimer. In contrast, the LPL aggregate was only poorly associated with ER chaperones, appearing to be trapped in an irreversible, inactive conformation destined for ER degradation.

The prosequence of human lactase-phlorizin hydrolase modulates the folding of the mature enzyme.

The efficient transport of proteins along the secretory pathway requires that the polypeptide adopts a stably folded conformation to egress the endoplasmic reticulum (ER). The transport-competent precursor of the brush border enzyme LPH, pro-LPH, undergoes an intracellular cleavage process in the trans-Golgi network between Arg(734) and Leu(735) to yield LPH beta(initial). The role of the prodomain comprising the N-terminally located 734 amino acids of pro-LPH, LPH alpha, in the folding events of LPH beta(initial) has been analyzed by the individual expression of both forms in COS-1 cells. Following synthesis at 37 degrees C LPH beta(initial) acquires a misfolded and enzymatically inactive conformation that is degraded by trypsin. A temperature shift to 20 degrees C generates a stable, trypsin-resistant, and enzymatically active LPH beta(initial) indicating that the individual expression of LPH beta(initial) results in a temperature-sensitive conformation. This form interacts at non-permissive temperatures sequentially with the ER chaperones immunoglobulin-binding protein and calnexin resulting in an ER retention. The LPH alpha prodomain resides in the ER when individually expressed. It reveals compact structural features that are stabilized by disulfide bridges. LPH alpha and LPH beta(initial) readily interact with each other upon coexpression, and this interaction appears to trigger the formation of a trypsin-resistant, correctly folded, enzymatically active, and transport-competent LPH beta(initial) polypeptide. These data clearly demonstrate that the proregion of pro-LPH is an intramolecular chaperone that is critically essential in facilitating the folding of the intermediate form LPH beta(initial) in the context of the pro-LPH polypeptide.

Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis.

Calreticulin and calnexin are Ca2+-binding proteins with chaperone activity in the endoplasmic reticulum. These proteins have been eliminated by gene replacement in Dictyostelium, the only microorganism known to harbor both proteins; family members in Dictyostelium are located at the base of phylogenetic trees. A dramatic decline in the rate of phagocytosis was observed in double mutants lacking calreticulin and calnexin, whereas only mild changes occurred in single mutants. Dictyostelium cells are professional phagocytes, capable of internalizing particles by a sequence of activities: adhesion of the particle to the cell surface, actin-dependent outgrowth of a phagocytic cup, and separation of the phagosome from the plasma membrane. In the double-null mutants, particles still adhered to the cell surface, but the outgrowth of phagocytic cups was compromised. Green fluorescent protein-tagged calreticulin and calnexin, expressed in wild-type cells, revealed a direct link of the endoplasmic reticulum to the phagocytic cup enclosing a particle, such that the Ca2+ storage capacity of calreticulin and calnexin might directly modulate activities of the actin system during particle uptake.