Involvement of lysosomal integral membrane protein-2 in the activation of autophagy.

Lysosomal integral membrane protein-2 (LIMP-2) is a type III transmembrane protein that is highly glycosylated and mainly localized to the lysosomal membrane. The diverse functions of LIMP-2 are currently being uncovered; however, its participation in macroautophagy, usually described as autophagy, has not yet been well-investigated. To determine the possible involvement of LIMP-2 in autophagic activity, we examined the intracellular amount of microtubule-associated protein 1 light chain 3 (LC3)-II, which is well-correlated with autophagosome levels, in exogenous rat LIMP-2-expressing COS7 and HEK293 cells. Transient or stable expression of LIMP-2-myc significantly increased the levels of LC3-II. Conversely, knockdown of LIMP-2 decreased the LC3-II levels in NIH3T3 cells. Furthermore, approaches using lysosomal protease inhibitors and mCherry-GFP-LC3 fluorescence suggested that exogenous expression of LIMP-2 increased the biogenesis of autophagosomes rather than decreased the lysosomal turnover of LC3-II. Considering the results of the biochemical assay and the quantitative fluorescence assay together, it is suggested that LIMP-2 has a possible involvement in autophagic activity, especially autophagosome biogenesis.

The Major Lysosomal Membrane Proteins LAMP-1 and LAMP-2 Participate in Differentiation of C2C12 Myoblasts.

Lysosomes are organelles that play a crucial role in the degradation of endocytosed molecules, phagocytosed macromolecules and autophagic substrates. The membrane of lysosomes contains several highly glycosylated membrane proteins, and lysosome-associated membrane protein (LAMP)-1 and LAMP-2 account for a major portion of the lysosomal membrane glycoproteins. Although it is well known that LAMP-2 deficiency causes Danon disease, which is characterized by cardiomyopathy, myopathy and mental retardation, the roles of lysosomal membrane proteins including LAMP-1 and LAMP-2 in myogenesis are not fully understood. In this study, to understand the role of LAMP proteins in the course of differentiation of myoblasts into myotubes, we used C2C12 myoblasts and found that the protein and mRNA levels of LAMP-1 and LAMP-2 were increased in the course of differentiation of C2C12 myoblasts into myotubes. Then, we investigated the effects of LAMP-1 or LAMP-2 knockdown on C2C12 myotube formation, and found that LAMP-1 or LAMP-2 depletion impaired the differentiation of C2C12 myoblasts and reduced the diameter of C2C12 myotubes. LAMP-2 knockdown more severely impaired C2C12 myotube formation compared with LAMP-1 knockdown, and knockdown of LAMP-1 did not exacerbate the suppressive effects of LAMP-2 knockdown on C2C12 myotube formation. In addition, knockdown of LAMP-1 or LAMP-2 decreased the expression levels of myogenic regulatory factors, MyoD and myogenin. These results demonstrate that both LAMP-1 and LAMP-2 are involved in C2C12 myotube formation and LAMP-2 may contribute dominantly to it.

ß-Taxilin participates in differentiation of C2C12 myoblasts into myotubes.

Myogenesis is required for the development of skeletal muscle. Accumulating evidence indicates that the expression of several genes are upregulated during myogenesis and these genes play pivotal roles in myogenesis. However, the molecular mechanism underlying myogenesis is not fully understood. In this study, we found that ß-taxilin, which is specifically expressed in the skeletal muscle and heart tissues, was progressively expressed during differentiation of C2C12 myoblasts into myotubes, prompting us to investigate the role of ß-taxilin in myogenesis. In C2C12 cells, knockdown of ß-taxilin impaired the fusion of myoblasts into myotubes, and decreased the diameter of myotubes. We also found that ß-taxilin interacted with dysbindin, a coiled-coil-containing protein. Knockdown of dysbindin conversely promoted the fusion of myoblasts into myotubes and increased the diameter of myotubes in C2C12 cells. Furthermore, knockdown of dysbindin attenuated the inhibitory effect of ß-taxilin depletion on myotube formation of C2C12 cells. These results demonstrate that ß-taxilin participates in myogenesis through suppressing the function of dysbindin to inhibit the differentiation of C2C12 myoblasts into myotubes.

Involvement of microRNA214 and transcriptional regulation in reductions in mevalonate pyrophosphate decarboxylase mRNA levels in stroke-prone spontaneously hypertensive rat livers.

Hypocholesterolemia has been epidemiologically identified as one of the causes of stroke (cerebral hemorrhage). We previously reported that lower protein levels of mevalonate pyrophosphate decarboxylase (MPD), which is responsible for reducing serum cholesterol levels in stroke-prone spontaneously hypertensive rats (SHRSP), in the liver were caused by a reduction in mRNA levels. However, the mechanism responsible for reducing MPD expression levels in the SHRSP liver remains unclear. Thus, we compared microRNA (miR)-214 combined with the 3'-untranslated region of MPD mRNA and heterogeneous nuclear RNA (hnRNA) between SHRSP and normotensive Wistar Kyoto rats (WKY). miR-214 levels in the liver were markedly higher in SHRSP than in WKY, whereas hnRNA levels were significantly lower. These results indicate that the upregulation of miR-214 and downregulation of MPD transcription in the liver both play a role in the development of hypocholesterolemia in SHRSP.

Comparative study of the steady-state subcellular distribution of lysosome-associated membrane glycoprotein-2 (LAMP-2) isoforms with GYXXΦ-type tyrosine-based motifs that interact differently with four adaptor protein (AP) complexes.

Lysosome-associated membrane protein-1 and -2 (LAMP-1 and LAMP-2, respectively) are type I transmembrane proteins. LAMP-2 comprises three splice isoforms (LAMP-2A, -B and-C) with different cytoplasmic tails (CTs). These three CTs possess different tyrosine-based motifs (GYXXΦ, where Φ is a bulky hydrophobic amino acid) at their C-termini. Interactions between tyrosine-based motifs and µ-subunits of four tetrameric adaptor protein (AP) complexes are necessary for their vesicular transport to lysosomes. Little is known about how the interaction strengths of these tyrosine motifs with µ-subunits affect the localization of isoforms to lysosomes. The interactions were first investigated using a yeast two-hybrid system to address this question. LAMP-2A-CT interacted with all four µ-subunits (µ1, µ2, µ3A and µ4 of AP-1, AP-2, AP-3 and AP-4, respectively). The interaction with µ3A was more robust than that with other µ-subunits. LAMP-2B-CT interacted exclusively and moderately with µ3A. LAMP-2C-CT did not detectably interact with any of the four µ-subunits. Immunofluorescence microscopy showed that all isoforms were localized in late endosomes and lysosomes. LAMP-2C was present in the plasma membrane and early endosomes; however, LAMP-2A and -2B were barely detectable in these organelles. In cell fractionation, LAMP-2A was the most abundant in the dense lysosomes, whereas LAMP-2C was significantly present in the low-density fraction containing the plasma membrane and early endosomes, in addition to the dense lysosomes. LAMP-2B considerably existed in the low-density late endosomal fraction. These data strongly suggest that the LAMP-2 isoforms are distributed differently in endocytic organelles depending on their interaction strengths with AP-3.

Comparison of receptors and enzymes regulating cholesterol levels in liver between SHR/NDmcr-cp rats and normotensive Wistar Kyoto rats at ten weeks of age.

The spontaneously hypertensive rat (SHR)/NDmcr-cp (SHR-cp), which is a metabolic syndrome model rat, was reported to show hypercholesteremia, as compared with lean littermates. The serum total cholesterol level in SHR-cp at 18 weeks of age is higher than that of normotensive Wistar Kyoto rat (WKY), but that in SHR-cp at 10 weeks of age is the same. The objective of this study is to clarify whether there are differences in the system regulating serum cholesterol levels between SHR-cp and WKY at 10 weeks of age. Total serum cholesterol levels, and cholesterol levels of high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) were similar in the two strains. However, the cholesterol levels in the liver of SHR-cp were lower than those of WKY. Next, mRNA levels of receptors (scavenger receptor class B type 1 [SRB1], LDL receptor [LDLR]) involved in uptake from serum to liver or enzymes of cholesterol catabolism (CYP7A1 and CYP8B1) and biosynthesis (mevalonate pyrophosphate decarboxylases [MPD]) in liver were compared between SHR-cp and WKY. High levels of MPD and LDLR and low levels of SRB1 were shown in SHR-cp, as compared with WKY. CYP7A1 and CYP8B1 levels were similar between SHR-cp and WKY. These results suggest that the serum cholesterol level in SHR-cp by the balance or regulation between the rise in cholesterol uptake and reduction in cholesterol biosynthesis in the liver is the same as that in WKY.

Effect of δ-tocotrienol on melanin content and enzymes for melanin synthesis in mouse melanoma cells.

In the present study, we investigated the dose-dependent effect of delta-tocotrienol long term (48, 72 h) on the melanin content of cells treated with delta-tocotrienol, and whether cells treated with delta-tocotrienol for long a time show cytotoxicity. We also examined whether other enzymes responsible for melanin biosynthesis, tyrosinase-related protein-1 (TRP-1) and -2 (TRP-2), are involved in the decrease in melanin levels. Protein levels in cells treated with 25 or 50 microM delta-tocotrienol for 48 h or 72 h were similar to those in control cells. Melanin content decreased by 44 (25 microM delta-tocotrienol) to 50% (50 microM) at 48 h, and by 14 to 21% at 72 h, compared to control levels. Tyrosinase activity, amounts of tyrosinase and TRP-1 decreased dependent on dose : by 50 (25 microM delta-tocotrienol) to 75% (50 microM), 20 to 45% and 42 to 82% at 48 h, and by 25 to 50%, 75 to 80% and 78 to 77% at 72 h, respectively. Although the amount of TRP-2 increased by 20% on treatment with 25 microM delta-tocotrienol for 48 h, it decreased by 52% on treatment with 50 microM delta-tocotrienol for 48 h. The amount of TRP-2 dose-dependently decreased by 55% and 75% on 72 h by treatment with 25 and 50 microM delta-tocotrienol, respectively. From these findings, delta-tocotrienol at up to 50 microM dose-dependently caused a reduction in melanin content by the decrease of TRP-1 and TRP-2 as well as tyrosinase, and no cytotoxicity.

High levels of oxidative stress exist in the brain than serum or kidneys in stroke-prone spontaneously hypertensive rats at ten weeks of age.

In the present study, we examined levels of oxidative stress in the serum, brain and kidneys of normotensive Wistar Kyoto rats (WKY) and stroke prone spontaneously hypertensive rats (SHRSP) at 10 weeks of age. Levels of advanced oxidation protein products (AOPP), oxidized albumin and oxidized proteins, markers of oxidative stress, were significantly decreased in serum among SHRSP as compared with WKY. Levels of oxidized proteins determined by immunoblotting were significantly increased in the brain, but not kidney, of SHRSP. The mRNA level of super oxide dismutase (SOD) determined by real time polymerase chain reaction (PCR) and the protein level of catalase assessed by immunoblotting were significantly increased in the brain of SHRSP. From these results, it was suggested that levels of oxidative stress were higher in the brain than serum or kidneys of SHRSP at 10 weeks of age, but are not caused by decreases in the expression of SOD and catalase.

The antioxidative and antilipidemic effects of different molecular weight chitosans in metabolic syndrome model rats.

The effect of high and low molecular weight chitosans (HMC; 1000 kDa, LMC; 30 kDa) on oxidative stress and hypercholesterolemia was investigated using male 6-week-old Wistar Kyoto rats as a normal model (Normal-rats) and spontaneously hypertensive rat/ND mcr-cp (SHP/ND) as a metabolic syndrome model (MS-rats), respectively. In Normal-rats, the ingestion of both chitosans over a 4 week period resulted in a significant decrease in total body weight (BW), glucose (Gl), triglyceride (TG), low density lipoprotein (LDL) and serum creatinine (Cre) levels. The ingestion of both chitosans also resulted in a lowered ratio of oxidized to reduced albumin and an increase in total plasma antioxidant activity. In addition to similar results in Normal-rats, the ingestion of only HMC over a 4 week period resulted in a significant decrease in total cholesterol levels in MS-rats. Further, the ingestion of LMC resulted in a significantly higher antioxidant activity than was observed for HMC in both rat models. In in vitro studies, LMC caused a significantly higher reduction in the levels of two stable radicals, compared to HMC, and the effect was both dose- and time-dependent. The findings also show that LDL showed strong binding in the case of HMC. These results suggest that LMC has a high antioxidant activity as well as antilipidemic effects, while HMC results in a significant reduction in the levels of pro-oxidants such as LDL in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress in the systemic circulation in metabolic model rats.

Tissue distribution of mevalonate pyrophosphate decarboxylase in guinea pig.

We previously reported that mevalonate pyrophosphate decarboxylase (MPD) is located in the cytosol and that MPD level in the liver is higher than in other rat tissues. In the present study, we further investigated the tissue distribution of MPD in guinea pigs by immunoblotting using anti-rat MPD antiserum. When immunoblot analysis was carried out using guinea pig brain, the antiserum reacted with 46-kDa protein as well as a substance with the same molecular weight of MPD in mice. Protein of 46-kDa detected in guinea pig liver treated with 0.1% pravastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor indicating a liver-specific effect, was increased 3-fold as compared with nontreated guinea pigs; however, 46-kDa protein in the brain treated with pravastatin was similar to that treated without pravastatin. When the subcellular distribution of MPD in the brain, liver, kidney, and testis, was examined by cell fractionation, MPD was mostly detected in the cytosol fraction of all tissues. From these data, the 46-kDa protein was identified as MPD. Next, when the tissue distribution of MPD was examined, MPD in the liver was higher than in other tissues. The relative amount of MPD in guinea pig kidney was higher than in rats and similar to in mice, as MPD in the liver of the same species was taken as 1. Furthermore, the correlation coefficient between guinea pigs and rats or mice in the tissue distribution of MPD was 0.69 or 0.72, respectively. These data indicate a relationship in tissue distribution between guinea pigs and rats or mice, although the tissue-specific regulator of MPD between species somewhat differed.

Decrease of cholesterol in mouse melanoma causes secretion of lysosomal enzymes.

We examined the change in the subcellular distribution of a lysosomal enzyme, beta-glucuronidase (beta-G), caused by decreased cholesterol levels in mouse melanoma cells using an HMG-CoA reductase inhibitor, lovastatin and lipoprotein-deficient serum (LDS). There was a decrease in the cholesterol content of the cells and increased secretion of the mature form of beta-G located in lysosomes, as documented by Percoll density gradient fractionation, digitonin permeabilization and immunoprecipitation. Furthermore, another lysosomal enzyme, cathepsin H, was found to be released in the medium from cells treated with lovastatin. Both the precursor and mature forms of cathepsin H were detected in the medium of treated cells. Next, when cells were treated with LDS without lovastatin, concomitantly with the decrease in the levels of cholesterol and beta-G activity in the cells, beta-G activity in the medium increased. Also, the ratio of beta-G (3.2-fold) released in the medium from cells treated with Dulbecco's modified Eagle medium (D-MEM) containing lovastatin and LDS was higher than that (2.3-fold) on treatment with D-MEM containing LDS without lovastatin. From these results, it was suggested that the exocytosis of mature enzymes from lysosomes into the medium or mis-sorting of the lysosomal precursor forms to the medium was caused by the lovastatin- and/or LDS-induced decrease in the cholesterol content of the cells, although the mechanism of secretion by lysosomal enzymes differed somewhat.

Effect of benzo[a]pyrene on P-glycoprotein-mediated transport in Caco-2 cell monolayer.

The main exposure pathway of benzo[a]pyrene (Bap) for humans is considered to be via the daily diet. The purpose of this study was to investigate the effect of BaP on the intestinal transport of chemicals mediated by P-glycoprotein (P-gp). The intestinal epithelial membrane transport of rhodamine-123 (Rho-123), a substrate of P-gp, was examined using a monolayer of the human Caco-2 cell line grown in transwells. In the monolayer exposed to Bap for 72 h before transport experiments, the ratio of the apparent permeability coefficients (P(app)) of Rho-123 efflux increased compared to that of the control. The permeability of rhodamine-B (Rho-B), not a substrate of P-gp, showed no difference between the monolayers. Treatment with quinidine or cyclosporine A, which are P-gp inhibitors, decreased the P(app) of Rho-123 to the same degree in both monolayers. The transport of Rho-123 was not influenced by the presence of Bap. Thus, Bap seemed not to act directly on the efflux activity of P-gp and be a binding site competitor of Rho-123. In the Caco-2 cells that enhanced the efflux of Rho-123 by the treatment with Bap, an increase in mRNA expression of MDR 1 (P-gp) was confirmed compared to that of control by RT-PCR. Furthermore, Western blot analysis using a monoclonal antibody, C219, demonstrated the increase of P-gp in Caco-2 cells exposed to Bap, compared with controls. It was inferred that Bap exposure induced the expression of P-gp, which led to the observed increase in efflux transport of Rho-123. The possibility was suggested that Bap might affect the disposition of medicines by increasing P-gp expression.

Chromatoid bodies: aggresome-like characteristics and degradation sites for organelles of spermiogenic cells.

We investigated the localization of several markers for lysosomes and aggresomes in the chromatoid bodies (CBs) by immunoelectron microscopy. We found so-called aggresomal markers such as Hsp70 and ubiquitin in the core of the CBs and vimentin and proteasome subunit around the CBs. Ubiquitin-conjugating enzyme (E2) was also found in the CBs. In tubulovesicular structures surrounding the CBs, lysosomal markers were detected but an endoplasmic reticulum retention signal (KDEL) was not. Moreover, proteins located in each subcellular compartment, including the cytosol, mitochondria, and nucleus, were detected in the CBs. Signals for cytochrome oxidase I (COXI) coded on mitochondrial DNA were also found in the CBs. Quantitative analysis of labeling density showed that all proteins examined were concentrated in the CBs to some extent. These results show that the CBs have some aggresomal features, suggesting that they are not a synthetic site as proposed previously but a degradation site where unnecessary DNA, RNA, and proteins are digested.

Constitutive expression of a COOH-terminal leucine mutant of lysosome-associated membrane protein-1 causes its exclusive localization in low density intracellular vesicles.

Lysosome-associated membrane protein-1 (LAMP-1) is a type I transmembrane protein with a short cytoplasmic tail that possesses a lysosome-targeting signal of GYQTI(382)-COOH. Wild-type (WT)-LAMP-1 was exclusively localized in high density lysosomes, and efficiency of LAMP-1's transport to lysosomes depends on its COOH-terminal amino acid residue. Among many different COOH-terminal amino acid substitution mutants of LAMP-1, a leucine-substituted mutant (I382L) displays the most efficient targeting to late endosomes and lysosomes [Akasaki et al. (2010) J. Biochem. 148: , 669-679]. In this study, we generated two human hepatoma cell lines (HepG2 cell lines) that stably express WT-LAMP-1 and I382L, and compared their intracellular distributions. The subcellular fractionation study using Percoll density gradient centrifugation revealed that WT-LAMP-1 had preferential localization in the high density secondary lysosomes where endogenous human LAMP-1 was enriched. In contrast, a major portion of I382L was located in a low density fraction. The low density fraction also contained approximately 80% of endogenous human LAMP-1 and significant amounts of endogenous ß-glucuronidase and LAMP-2, which probably represents occurrence of low density lysosomes in the I382L-expressing cells. Double immunofluorescence microscopic analyses distinguished I382L-containing intracellular vesicles from endogenous LAMP-1-containing lysosomes and early endosomes. Altogether, constitutive expression of I382L causes its aberrant intracellular localization and generation of low density lysosomes, indicating that the COOH-terminal isoleucine is critical for normal localization of LAMP-1 in the dense lysosomes.

COOH-terminal isoleucine of lysosome-associated membrane protein-1 is optimal for its efficient targeting to dense secondary lysosomes.

Lysosome-associated membrane protein-1 (LAMP-1) consists of a highly glycosylated luminal domain, a single-transmembrane domain and a short cytoplasmic tail that possesses a lysosome-targeting signal (GYQTI(382)) at the COOH terminus. It is hypothesized that the COOH-terminal isoleucine, I(382), could be substituted with any other bulky hydrophobic amino acid residue for LAMP-1 to exclusively localize in lysosomes. In order to test this hypothesis, we compared subcellular distribution of four substitution mutants with phenylalanine, leucine, methionine and valine at the COOH-terminus (termed I382F, I382L, I382M and I382V, respectively) with that of wild-type (WT)-LAMP-1. Double-labelled immunofluorescence analyses showed that these substitution mutants were localized as significantly to late endocytic organelles as WT-LAMP-1. However, the quantitative subcellular fractionation study revealed different distribution of WT-LAMP-1 and these four COOH-terminal mutants in late endosomes and dense secondary lysosomes. WT-LAMP-1 was accumulated three to six times more in the dense lysosomal fraction than the four mutants. The level of WT-LAMP-1 in late endosomal fraction was comparable to those of I382F, I382M and I382V. Conversely, I382L in the late endosomal fraction was approximately three times more abundant than WT-LAMP-1. These findings define the presence of isoleucine residue at the COOH-terminus of LAMP-1 as critical in governing its efficient delivery to secondary lysosomes and its ratio of lysosomes to late endosomes.

Antioxidant properties of some different molecular weight chitosans.

Chitosan, a cationic polysaccharide, is widely employed as dietary supplement and in pharmacological and biomedical applications. Although numerous studies have focused on its applications as pharmaceutical excipients or bioactive reagents, relationships between molecular weight (Mr) and biological properties remain unclear. The focus of this study was on the antioxidant properties of several Mr chitosans. We measured the ability of seven Mr chitosans (CT1; 2.8 kDa, CT2; 17.0 kDa, CT3; 33.5 kDa, CT4; 62.6 kDa, CT5; 87.7 kDa, CT6; 604 kDa, CT7; 931 kDa) to protect plasma protein from oxidation by peroxyl radicals derived from 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH). A comparison of the antioxidant action of high Mr chitosans (CT6-CT7) with that of low Mr chitosans (CT1-CT5) showed that low Mr chitosans (CT1-CT5) were more effective in preventing the formation of carbonyl groups in plasma protein exposed to peroxyl radicals. AAPH substantially increases plasma protein carbonyl content via the oxidation of human serum albumin (HSA). We also measured the ability of these chitosans to protect HSA against oxidation by AAPH. Low Mr chitosans (CT1-CT5) were found to effectively prevent the formation of carbonyl groups in HSA, when exposed to peroxyl radicals. Low Mr chitosans were also good scavengers of N-centered radicals, but high Mr chitosans were much less effective. We also found a strong correlation between antioxidant activity and the Mr of chitosans in vitro. These activities were also determined by using the 'TPAC' test. These results suggest that low Mr chitosans (CT1-CT3) may be absorbed well from the gastrointestinal tract and inhibit neutrophil activation and oxidation of serum albumin that is frequently observed in patients plasma undergoing hemodialysis, resulting in a reduction in oxidative stress associated with uremia.

Dendritic cell-lysosomal-associated membrane protein (LAMP) and LAMP-1-HIV-1 gag chimeras have distinct cellular trafficking pathways and prime T and B cell responses to a diverse repertoire of epitopes.

Ag processing is a critical step in defining the repertoire of epitope-specific immune responses. In the present study, HIV-1 p55Gag Ag was synthesized as a DNA plasmid with either lysosomal-associated membrane protein-1 (LAMP/gag) or human dendritic cell-LAMP (DC-LAMP/gag) and used to immunize mice. Analysis of the cellular trafficking of these two chimeras demonstrated that both molecules colocalized with MHC class II molecules but differed in their overall trafficking to endosomal/lysosomal compartments. Following DNA immunization, both chimeras elicited potent Gag-specific T and B cell immune responses in mice but differ markedly in their IL-4 and IgG1/IgG2a responses. The DC-LAMP chimera induced a stronger Th type 1 response. ELISPOT analysis of T cell responses to 122 individual peptides encompassing the entire p55gag sequence (15-aa peptides overlapping by 11 residues) showed that DNA immunization with native gag, LAMP/gag, or DC-LAMP/gag induced responses to identical immunodominant CD4+ and CD8+ peptides. However, LAMP/gag and DC-LAMP/gag plasmids also elicited significant responses to 23 additional cryptic epitopes that were not recognized after immunization with native gag DNA. The three plasmids induced T cell responses to a total of 39 distinct peptide sequences, 13 of which were induced by all three DNA constructs. Individually, DC-LAMP/gag elicited the most diverse response, with a specific T cell response against 35 peptides. In addition, immunization with LAMP/gag and DC-LAMP/gag chimeras also promoted Ab secretion to an increased number of epitopes. These data indicate that LAMP-1 and DC-LAMP Ag chimeras follow different trafficking pathways, induce distinct modulatory immune responses, and are able to present cryptic epitopes.

Disruptive effect of chloroquine on lysosomes in cultured rat hepatocytes.

Chloroquine has been used as an anti-malarial drug and is known as a lysosomotropic amine as well. The effects of chloroquine on lysosomal integrity in cultured rat hepatocytes were studied by measuring lysosomal enzyme beta-glucuronidase (beta-G) or lysosomal membrane glycoprotein (lamp-1) in Percoll density gradient fractions, in the cytosolic fraction obtained from cells permeabilized by digitonin or in the cytosolic fraction obtained by conventional cell fractionation. The distribution of beta-G on a Percoll density gradient in chloroquine-treated cells was approximately similar to that of a cytosolic protein, mevalonate pyrophosphate decarboxylase, in nontreated cells. Lamp-1 was decreased in the lysosomal fractions on a Percoll density gradient in chloroquine-treated cells, and was increased in the plasma membrane fraction, as compared with the levels in nontreated cells. Furthermore, after cells were cultured in the presence and absence of chloroquine, the proportions of beta-G activity in the cytosolic fraction obtained from the digitonin-permeabilized cells were 19% and 4%, while those in the cytosolic fraction obtained by conventional cell fractionation were 54% and 26%, respectively. From these findings, we infer that chloroquine caused the disruption of lysosomes in the living cells, and that lysosomes treated with chloroquine were easily disrupted by homogenization or centrifugation during cell fractionation.

Ile (476), a constituent of di-leucine-based motif of a major lysosomal membrane protein, LGP85/LIMP II, is important for its proper distribution in late endosomes and lysosomes.

Lysosomal membrane glycoprotein termed LGP85 or LIMP II extends a COOH-terminal cytoplasmic tail of R459GQGSMDEGTADERAPLIRT478, in which an L475 I476 sequence lies as a di-leucine-based motif for lysosomal targeting. In the present study, we explored the role of the I476 residue in the localization of LGP85 to the endocytic organelles using two substitution mutants called I476A and I476L in which alanine and leucine are replaced at I476, respectively, and I476R477T478-deleted LGP85 called Delta 476-478. Immunofluorescence analyses showed that I476A and I476L are largely colocalized in intracellular organelles with an endogenous late endosomal and lysosomal marker, LAMP-1, but there were some granules in which staining for the LGP85 mutants was prominent, while Delta 476-478 is detected in LAMP-1-positive and LAMP-1-negative intracellular organelles, and on the cell surface. The subcellular fractionation studies revealed that I476A, I476L, and Delta 476-478 are different from wild-type LGP85 in the distribution of early endosomes, late endosomes, and lysosomes. I476A and I476L are present more in late endosomes than in the densest lysosomes, whereas wild-type LGP85 is mainly lysosomal. Substitution of I476 for A and L differentially modified the ratios of late endosomal to lysosomal LGP85. A major portion of Delta 476-478 resided in the light buoyant density fraction containing plasma membrane and early endosomes. Taken together, these results indicate that the existence of the 476th amino acid residue is essential for localization of LGP85 to late endocytic compartments. The fact that isoleucine but not leucine is in the 476th position is especially of importance in the proper distribution of LGP85 in late endosomes and lysosomes.

Subcellular distribution of mouse mevalonate pyrophosphate decarboxylase.

Mevalonate pyrophosphate decarboxylase (MPD) is considered to be a cytosolic protein. Recently, other groups reported that MPD is mostly located in the peroxisomes. In this study, we examined whether the expression of MPD in mice depends on the proliferation of peroxisomes, and whether MPD is predominantly located in the peroxisomes or the cytosol of mice. No increase in the protein level of MPD was observed in the crude extract of the livers of mice administered with peroxisome proliferative drugs. The result suggests that the expression of MPD is independent of the proliferation of peroxisomes, and may be maintained via a specific regulatory mechanism, different from the regulation of the expression of peroxisome proliferator-activated receptor alpha. When the subcellular distribution of MPD in mouse melanoma (B16F10) cells was examined by cell fractionation, MPD was detected in the cytosol of B16F10 cells, but not in the peroxisomes. In permeabilized B16F10 cells treated with digitonin, which lack cytosolic enzymes, 80% and 20% of MPD, 75% and 25% of lactate dehydrogenase, or 2% and 98% of catalase, existed in the medium and in the cell, respectively. From these results, it indicated that MPD was predominantly located in the cytosol and did not exist in the peroxisomes of B16F10 cells.