Role for LAMP-2 in endosomal cholesterol transport.

The mechanisms of endosomal and lysosomal cholesterol traffic are still poorly understood. We showed previously that unesterified cholesterol accumulates in the late endosomes and lysosomes of fibroblasts deficient in both lysosome associated membrane protein-2 (LAMP-2) and LAMP-1, two abundant membrane proteins of late endosomes and lysosomes. In this study we show that in cells deficient in both LAMP-1 and LAMP-2 (LAMP(-/-)), low-density lipoprotein (LDL) receptor levels and LDL uptake are increased as compared to wild-type cells. However, there is a defect in esterification of both endogenous and LDL cholesterol. These results suggest that LAMP(-/-) cells have a defect in cholesterol transport to the site of esterification in the endoplasmic reticulum, likely due to defective export of cholesterol out of late endosomes or lysosomes. We also show that cholesterol accumulates in LAMP-2 deficient liver and that overexpression of LAMP-2 retards the lysosomal cholesterol accumulation induced by U18666A. These results point to a critical role for LAMP-2 in endosomal/lysosomal cholesterol export. Moreover, the late endosomal/lysosomal cholesterol accumulation in LAMP(-/-) cells was diminished by overexpression of any of the three isoforms of LAMP-2, but not by LAMP-1. The LAMP-2 luminal domain, the membrane-proximal half in particular, was necessary and sufficient for the rescue effect. Taken together, our results suggest that LAMP-2, its luminal domain in particular, plays a critical role in endosomal cholesterol transport and that this is distinct from the chaperone-mediated autophagy function of LAMP-2.

Impaired phagosomal maturation in neutrophils leads to periodontitis in lysosomal-associated membrane protein-2 knockout mice.

Inflammatory periodontal diseases constitute one of the most common infections in humans, resulting in the destruction of the supporting structures of the dentition. Circulating neutrophils are an essential component of the human innate immune system. We observed that mice deficient for the major lysosomal-associated membrane protein-2 (LAMP-2) developed severe periodontitis early in life. This development was accompanied by a massive accumulation of bacterial plaque along the tooth surfaces, gingival inflammation, alveolar bone resorption, loss of connective tissue fiber attachment, apical migration of junctional epithelium, and pathological movement of the molars. The inflammatory lesions were dominated by polymorphonuclear leukocytes (PMNs) apparently being unable to efficiently clear bacterial pathogens. Systemic treatment of LAMP-2-deficient mice with antibiotics prevented the periodontal pathology. Isolated PMNs from LAMP-2-deficient mice showed an accumulation of autophagic vacuoles and a reduced bacterial killing capacity. Oxidative burst response was not altered in these cells. Latex bead and bacterial feeding experiments showed a reduced ability of the phagosomes to acquire an acidic pH and late endocytic markers, suggesting an impaired fusion of late endosomes-lysosomes with phagosomes. This study underlines the importance of LAMP-2 for the maturation of phagosomes in PMNs. It also underscores the requirement of lysosomal fusion events to provide sufficient antimicrobial activity in PMNs, which is needed to prevent periodontal disease.

Arrested maturation of Neisseria-containing phagosomes in the absence of the lysosome-associated membrane proteins, LAMP-1 and LAMP-2.

Mature, microbicidal phagosomes are rich in the lysosome-associated membrane proteins, LAMP-1 and LAMP-2, two highly glycosylated proteins presumed to form a protective barrier lining the phagosomal membrane. Pathogenic Neisseria secrete a protease that selectively cleaves LAMP-1, suggesting a critical role for LAMP proteins in the microbicidal competence of phagosomes. To determine the requirement for LAMP proteins in bacterial phagocytosis, we employed embryonic fibroblasts isolated from knockout mice lacking lamp-1, lamp-2 or both genes, as well as small interfering RNA (siRNA)-mediated knockdown of LAMP expression in a human epithelial cell line. Like wild-type cells, those lacking either LAMP-1 or LAMP-2 alone formed phagosomes that gradually acquired microbicidal activity and curtailed bacterial growth. In contrast, LAMP-1 and LAMP-2 double-deficient fibroblasts failed to kill engulfed Neisseria gonorrhoeae. In these cells, maturation was arrested prior to the acquisition of Rab7. As a result, the Rab7-interacting lysosomal protein (RILP, a Rab7 effector) was not recruited to the phagosomes, which were consequently unable to undergo dynein/dynactin-mediated centripetal displacement along microtubules and remained in a predominantly peripheral location. The inability of such phagosomes to migrate towards lysosomes likely contributed to their incomplete maturation and inability to eliminate bacteria. These findings suggest that neisserial degradation of LAMP-1 is not sufficient to affect its survival within the phagosome, and establish LAMP proteins as critical components in the process whereby phagosomes acquire microbicidal capabilities.

ATP binding to the KTN/RCK subunit KtrA from the K+ -uptake system KtrAB of Vibrio alginolyticus: its role in the formation of the KtrAB complex and its requirement in vivo.

Subunit KtrA of the bacterial Na(+)-dependent K(+)-translocating KtrAB systems belongs to the KTN/RCK family of regulatory proteins and protein domains. They are located at the cytoplasmic side of the cell membrane. By binding ligands they regulate the activity of a number of K(+) transporters and K(+) channels. To investigate the function of KtrA from the bacterium Vibrio alginolyticus (VaKtrA), the protein was overproduced in His-tagged form (His(10)-VaKtrA) and isolated by affinity chromatography. VaKtrA contains a G-rich, ADP-moiety binding beta-alpha-beta-fold ("Rossman fold"). Photocross-linking and flow dialysis were used to determine the binding of [(32)P]ATP and [(32)P]NAD(+) to His(10)-VaKtrA. Binding of other nucleotides was estimated from the competition by these compounds of the binding of the (32)P-labeled nucleotides to the protein. [gamma-(32)P]ATP bound with high affinity to His(10)-VaKtrA (K(D) of 9 microm). All other nucleotides tested exhibited K(D) (K(i)) values of 30 microm or higher. Limited proteolysis with trypsin showed that ATP was the only nucleotide that changed the conformation of VaKtrA. ATP specifically promoted complex formation of VaKtrA with the His-tagged form of its K(+)-translocating partner, VaKtrB-His(6), as detected both in an overlay experiment and in an experiment in which VaKtrA was added to VaKtrB-His(6) bound to Ni(2+)-agarose. In intact cells of Escherichia coli both a high of membrane potential and a high cytoplasmic ATP concentration were required for VaKtrAB activity. C-terminal deletions in VaKtrA showed that for in vivo activity at least 169 N-terminal amino acid residues of its total of 220 are required and that its 40 C-terminal residues are dispensable.

Mannose 6-phosphate receptors, Niemann-Pick C2 protein, and lysosomal cholesterol accumulation.

Niemann-Pick disease type C (NPC), caused by mutations in the NPC1 gene or the NPC2 gene, is characterized by the accumulation of unesterified cholesterol and other lipids in endo/lysosomal compartments. NPC2 is a small, soluble, lysosomal protein that is targeted to this compartment via a mannose 6-phosphate-inhibitable pathway. To obtain insight into the roles of mannose 6-phosphate receptors (MPRs) in NPC2 targeting, we here examine the trafficking and function of NPC2 in fibroblast lines deficient in one or both of the two MPRs, MPR46 and MPR300. We demonstrate that either MPR alone is sufficient to transport NPC2 to the endo/lysosomal compartment, although MPR300 seems to be more efficient than MPR46. In the absence of both MPRs, NPC2 is secreted into the culture medium, and only a small amount of intracellular NPC2 can be detected, mainly in the endoplasmic reticulum. This leads to massive accumulation of unesterified cholesterol in the endo/lysosomal compartment of the MPR46/300-deficient fibroblasts, a phenotype similar to that of the NPC patient fibroblasts. In addition, we observed an upregulation of NPC1 protein and mRNA in the MPR-double-deficient cells. Taken together, our results suggest that the lysosomal targeting of NPC2 is strictly dependent on MPRs in fibroblasts.

Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/LAMP-2 double-deficient fibroblasts.

Mice double deficient in LAMP-1 and -2 were generated. The embryos died between embryonic days 14.5 and 16.5. An accumulation of autophagic vacuoles was detected in many tissues including endothelial cells and Schwann cells. Fibroblast cell lines derived from the double-deficient embryos accumulated autophagic vacuoles and the autophagy protein LC3II after amino acid starvation. Lysosomal vesicles were larger and more peripherally distributed and showed a lower specific density in Percoll gradients in double deficient when compared with control cells. Lysosomal enzyme activities, cathepsin D processing and mannose-6-phosphate receptor expression levels were not affected by the deficiency of both LAMPs. Surprisingly, LAMP-1 and -2 deficiencies did not affect long-lived protein degradation rates, including proteolysis due to chaperone-mediated autophagy. The LAMP-1/2 double-deficient cells and, to a lesser extent, LAMP-2 single-deficient cells showed an accumulation of unesterified cholesterol in endo/lysosomal, rab7, and NPC1 positive compartments as well as reduced amounts of lipid droplets. The cholesterol accumulation in LAMP-1/2 double-deficient cells could be rescued by overexpression of murine LAMP-2a, but not by LAMP-1, highlighting the more prominent role of LAMP-2. Taken together these findings indicate partially overlapping functions for LAMP-1 and -2 in lysosome biogenesis, autophagy, and cholesterol homeostasis.