NPC1 plays a role in the trafficking of specific cargo to melanosomes.

Niemann-Pick type C1 (NPC1) protein is a multimembrane spanning protein of the lysosome limiting membrane that facilitates intracellular cholesterol and sphingolipid transport. Loss-of-function mutations in the NPC1 protein cause Niemann-Pick disease type C1, a lysosomal storage disorder characterized by the accumulation of cholesterol and sphingolipids within lysosomes. To investigate whether the NPC1 protein could also play a role in the maturation of the endolysosomal pathway, here, we have investigated its role in a lysosome-related organelle, the melanosome. Using a NPC1-KO melanoma cell model, we found that the cellular phenotype of Niemann-Pick disease type C1 is associated with a decreased pigmentation accompanied by low expression of the melanogenic enzyme tyrosinase. We propose that the defective processing and localization of tyrosinase, occurring in the absence of NPC1, is a major determinant of the pigmentation impairment in NPC1-KO cells. Along with tyrosinase, two other pigmentation genes, tyrosinase-related protein 1 and Dopachrome-tautomerase have lower protein levels in NPC1 deficient cells. In contrast with the decrease in pigmentation-related protein expression, we also found a significant intracellular accumulation of mature PMEL17, the structural protein of melanosomes. As opposed to the normal dendritic localization of melanosomes, the disruption of melanosome matrix generation in NPC1 deficient cells causes an accumulation of immature melanosomes adjacent to the plasma membrane. Together with the melanosomal localization of NPC1 in WT cells, these findings suggest that NPC1 is directly involved in tyrosinase transport from the trans-Golgi network to melanosomes and melanosome maturation, indicating a novel function for NPC1.

Inhibition of the Niemann-Pick C1 protein is a conserved feature of multiple strains of pathogenic mycobacteria.

Mycobacterium tuberculosis (Mtb) survives and replicates within host macrophages (MΦ) and subverts multiple antimicrobial defense mechanisms. Previously, we reported that lipids shed by pathogenic mycobacteria inhibit NPC1, the lysosomal membrane protein deficient in the lysosomal storage disorder Niemann-Pick disease type C (NPC). Inhibition of NPC1 leads to a drop in lysosomal calcium levels, blocking phagosome-lysosome fusion leading to mycobacterial survival. We speculated that the production of specific cell wall lipid(s) that inhibit NPC1 could have been a critical step in the evolution of pathogenicity. We therefore investigated whether lipid extracts from clinical Mtb strains from multiple Mtb lineages, Mtb complex (MTBC) members and non-tubercular mycobacteria (NTM) inhibit the NPC pathway. We report that inhibition of the NPC pathway was present in all clinical isolates from Mtb lineages 1, 2, 3 and 4, Mycobacterium bovis and the NTM, Mycobacterium abscessus and Mycobacterium avium. However, lipid extract from Mycobacterium canettii, which is considered to resemble the common ancestor of the MTBC did not inhibit the NPC1 pathway. We conclude that the evolution of NPC1 inhibitory mycobacterial cell wall lipids evolved early and post divergence from Mycobacterium canettii-related mycobacteria and that this activity contributes significantly to the promotion of disease.

Anti-TNF therapy for inflammatory bowel disease in patients with neurodegenerative Niemann-Pick disease Type C.

Background:  Blockade of tumour necrosis factor (anti-TNF) is effective in patients with Crohn's Disease but has been associated with infection risk and neurological complications such as demyelination. Niemann-Pick disease Type C1 (NPC1) is a lysosomal storage disorder presenting in childhood with neurological deterioration, liver damage and respiratory infections. Some NPC1 patients develop severe Crohn's disease. Our objective was to investigate the safety and effectiveness of anti-TNF in NPC1 patients with Crohn's disease. Methods: Retrospective data on phenotype and therapy response were collected in 2019-2020 for the time period 2014 to 2020 from patients in the UK, France, Germany and Canada with genetically confirmed NPC1 defects and intestinal inflammation. We investigated TNF secretion in peripheral blood mononuclear cells treated with NPC1 inhibitor in response to bacterial stimuli . Results: NPC1 inhibitor treated peripheral blood mononuclear cells (PBMCs) show significantly increased TNF production after lipopolysaccharide or bacterial challenge providing a rationale for anti-TNF therapy. We identified 4 NPC1 patients with Crohn's disease (CD)-like intestinal inflammation treated using anti-TNF therapy (mean age of onset 8.1 years, mean treatment length 27.75 months, overall treatment period 9.25 patient years). Anti-TNF therapy was associated with reduced gastrointestinal symptoms with no apparent adverse neurological events. Therapy improved intestinal inflammation in 4 patients. Conclusions: Anti-TNF therapy appears safe in patients with NPC1 and is an effective treatment strategy for the management of intestinal inflammation in these patients.

Defective iron homeostasis and hematological abnormalities in Niemann-Pick disease type C1.

Background: Niemann-Pick disease type C1 (NPC1) is a neurodegenerative lysosomal storage disorder characterized by the accumulation of multiple lipids in the late endosome/lysosomal system and reduced acidic store calcium. The lysosomal system regulates key aspects of iron homeostasis, which prompted us to investigate whether there are hematological abnormalities and iron metabolism defects in NPC1. Methods: Iron-related hematological parameters, systemic and tissue metal ion and relevant hormonal and proteins levels, expression of specific pro-inflammatory mediators and erythrophagocytosis were evaluated in an authentic mouse model and in a large cohort of NPC patients. Results: Significant changes in mean corpuscular volume and corpuscular hemoglobin were detected in Npc1 -/- mice from an early age. Hematocrit, red cell distribution width and hemoglobin changes were observed in late-stage disease animals. Systemic iron deficiency, increased circulating hepcidin, decreased ferritin and abnormal pro-inflammatory cytokine levels were also found. Furthermore, there is evidence of defective erythrophagocytosis in Npc1 -/- mice and in an in vitro NPC1 cellular model. Comparable hematological changes, including low normal serum iron and transferrin saturation and low cerebrospinal fluid ferritin were confirmed in NPC1 patients. Conclusions: These data suggest loss of iron homeostasis and hematological abnormalities in NPC1 may contribute to the pathophysiology of this disease.

Unbiased yeast screens identify cellular pathways affected in Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is a rare lysosomal storage disease caused by mutations in either the NPC1 or NPC2 genes. Mutations in the NPC1 gene lead to the majority of clinical cases (95%); however, the function of NPC1 remains unknown. To gain further insights into the biology of NPC1, we took advantage of the homology between the human NPC1 protein and its yeast orthologue, Niemann-Pick C-related protein 1 (Ncr1). We recreated the NCR1 mutant in yeast and performed screens to identify compensatory or redundant pathways that may be involved in NPC pathology, as well as proteins that were mislocalized in NCR1-deficient yeast. We also identified binding partners of the yeast Ncr1 orthologue. These screens identified several processes and pathways that may contribute to NPC pathogenesis. These included alterations in mitochondrial function, cytoskeleton organization, metal ion homeostasis, lipid trafficking, calcium signalling, and nutrient sensing. The mitochondrial and cytoskeletal abnormalities were validated in patient cells carrying mutations in NPC1, confirming their dysfunction in NPC disease.

Transcriptional activation of RagD GTPase controls mTORC1 and promotes cancer growth.

The mechanistic target of rapamycin complex 1 (mTORC1) is recruited to the lysosome by Rag guanosine triphosphatases (GTPases) and regulates anabolic pathways in response to nutrients. We found that MiT/TFE transcription factors-master regulators of lysosomal and melanosomal biogenesis and autophagy-control mTORC1 lysosomal recruitment and activity by directly regulating the expression of RagD. In mice, this mechanism mediated adaptation to food availability after starvation and physical exercise and played an important role in cancer growth. Up-regulation of MiT/TFE genes in cells and tissues from patients and murine models of renal cell carcinoma, pancreatic ductal adenocarcinoma, and melanoma triggered RagD-mediated mTORC1 induction, resulting in cell hyperproliferation and cancer growth. Thus, this transcriptional regulatory mechanism enables cellular adaptation to nutrient availability and supports the energy-demanding metabolism of cancer cells.

Impaired antibacterial autophagy links granulomatous intestinal inflammation in Niemann-Pick disease type C1 and XIAP deficiency with NOD2 variants in Crohn's disease.

OBJECTIVE: Patients with Niemann-Pick disease type C1 (NPC1), a lysosomal lipid storage disorder that causes neurodegeneration and liver damage, can present with IBD, but neither the significance nor the functional mechanism of this association is clear. We studied bacterial handling and antibacterial autophagy in patients with NPC1. DESIGN: We characterised intestinal inflammation in 14 patients with NPC1 who developed IBD. We investigated bacterial handling and cytokine production of NPC1 monocytes or macrophages in vitro and compared NPC1-associated functional defects to those caused by IBD-associated nucleotide-binding oligomerization domain-containing protein 2 (NOD2) variants or mutations in X-linked inhibitor of apoptosis (XIAP). RESULTS: Patients with the lysosomal lipid storage disorder NPC1 have increased susceptibility to early-onset fistulising colitis with granuloma formation, reminiscent of Crohn's disease (CD). Mutations in NPC1 cause impaired autophagy due to defective autophagosome function that abolishes NOD2-mediated bacterial handling in vitro similar to variants in NOD2 or XIAP deficiency. In contrast to genetic NOD2 and XIAP variants, NPC1 mutations do not impair NOD2-receptor-interacting kinase 2 (RIPK2)-XIAP-dependent cytokine production. Pharmacological activation of autophagy can rescue bacterial clearance in macrophages in vitro by increasing the autophagic flux and bypassing defects in NPC1. CONCLUSIONS: NPC1 confers increased risk of early-onset severe CD. Our data support the concept that genetic defects at different checkpoints of selective autophagy cause a shared outcome of CD-like immunopathology linking monogenic and polygenic forms of IBD. Muramyl dipeptide-driven cytokine responses and antibacterial autophagy induction are parallel and independent signalling cascades downstream of the NOD2-RIPK2-XIAP complex.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway.

BACKGROUND: Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. METHODS: The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. RESULTS: Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. CONCLUSION: These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

Immune dysfunction in Niemann-Pick disease type C.

Lysosomal storage diseases are inherited monogenic disorders in which lysosome function is compromised. Although individually very rare, they occur at a collective frequency of approximately one in five thousand live births and usually have catastrophic consequences for health. The lysosomal storage diseases Niemann-Pick disease type C (NPC) is caused by mutations predominantly in the lysosomal integral membrane protein NPC1 and clinically presents as a progressive neurodegenerative disorder. In this article we review data that demonstrate significant dysregulation of innate immunity in NPC, which occurs both in peripheral organs and the CNS. In particular pro-inflammatory responses promote disease progression and anti-inflammatory drugs provide benefit in animal models of the disease and are an attractive target for clinical intervention in this disorder. Niemann-Pick disease type C is a rare, devastating, inherited lysosomal storage disease with a unique cellular phenotype characterized by lysosomal accumulation of sphingosine, various glycosphingolipids and cholesterol and a reduction in lysosomal calcium. In this review we highlight the impact of the disease on innate immune activities in both the central nervous system (CNS) and peripheral tissues and discuss their contributions to pathology and the underlying mechanisms.

Expression of Ca²âº-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells.

The second messenger NAADP triggers Ca(2+) release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2(-/-)), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca(2+) responses as assessed by single-cell Ca(2+) imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2(-/-) cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca(2+)-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca(2+) release. High-affinity [(32)P]NAADP binding still occurs in Tpcn1/2(-/-) tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca(2+)-permeable channels indispensable for NAADP signalling.

Measuring the phagocytic activity of cells.

Phagocytosis is a critical biological activity through which the host can protect itself from infectious and non-infectious environmental particles and remove unwanted host cells in order to maintain tissue homeostasis. Phagocytosis is an ancient, conserved process that is apparent in all multicellular organisms. The process of phagocytosis requires the recognition of ligands on particles by specific receptors expressed by the phagocyte that promote internalization via reorganization of cytoskeletal elements and directed formation of the phagosome. Subsequent phagosome-lysosome fusion delivers the contents for destruction and recycling in the acidic compartment. Significantly, receptor engagement and uptake can also trigger intracellular signaling pathways that initiate appropriate innate immune and pro-inflammatory or anti-inflammatory responses dependent upon the nature of the particle. The important benefits of phagocytosis to host survival are exemplified by the detrimental effects to health that occur when phagocytic efficiency is diminished. In an overview, we discuss the different experimental approaches or options that can be considered when investigating and determining the characteristics and quantification of phagocytic activity. These criteria will include choice of phagocytic cell type, selection, and method of labeling of particle for monitoring internalization, targeting of particles to specific receptors, and quantification of ingestion either at the single cell or at the population level. We provide two detailed examples of phagocytosis assays.

Innate immune responses in the brain of sphingolipid lysosomal storage diseases.

Lysosomal storage diseases (LSDs) are mainly caused by the defective activity of lysosomal hydrolases. A sub-class of LSDs are the sphingolipidoses, in which sphingolipids accumulate intra-cellularly. We here discuss the role of innate immunity in the sphingolipidoses, and compare the pathways of activation in two classical sphingolipidoses, namely Gaucher disease and Sandhoff disease, and in Niemann-Pick C disease, in which the main storage material is cholesterol but sphingolipids also accumulate. We discuss the mechanisms leading to neuroinflammation, and the different pathways of neuroinflammation in the different diseases, and suggest that intervention in these pathways may be a useful therapeutic approach to address these devastating human diseases.

Systemic inflammation switches the inflammatory cytokine profile in CNS Wallerian degeneration.

Axon loss in the CNS is characteristic of many neurodegenerative diseases but the mechanisms of axon degeneration are poorly understood. In particular, we know little of the inflammatory response triggered by CNS axon degeneration with comparison to that provoked by death of the neuronal cell body. We show that Wallerian degeneration of the mouse optic nerve induces transcription of TGF-beta1 and TNF-alpha, but not pro-inflammatory cytokines IL-1beta and IL-6 and microglial activation. This atypical inflammatory response resembles macrophages that have phagocytosed apoptotic cells and prion-infected CNS. Significantly, peripheral endotoxin challenge after injury switched this profile by inducing IL-1beta, IL-6 transcripts, other inflammation-associated products and reducing neurofilament immunoreactivity. We propose that microglia are activated by Wallerian degeneration and persist in an atypical but "primed" state and can be switched by systemic inflammation to provoke a classical pro-inflammatory profile with potentially deleterious consequences.

Sulfated glycosphingolipid as mediator of phagocytosis: SM4s enhances apoptotic cell clearance and modulates macrophage activity.

Sulfoglycolipids are present on the surface of a variety of cells. The sulfatide SM4s is increased in lung, renal, and colon cancer and is associated with an adverse prognosis, possibly due to a low immunoreactivity of the tumor. As macrophages significantly contribute to the inflammatory infiltrate in malignancies, we postulated that SM4s may modulate macrophage function. We have investigated the effect of SM4s on the uptake of apoptotic tumor cells, macrophage cytokine profile, and receptor expression. Using flow cytometry and microscopic analyses, we found that coating apoptotic murine carcinoma cells from the colon and kidney with SM4s promoted their phagocytosis by murine macrophages up to 3-fold ex vivo and in vivo. This increased capacity was specifically inhibited by preincubation of macrophages with oxidized or acetylated low density lipoprotein and maleylated albumin, indicating involvement of scavenger receptors in this interaction. The uptake of SM4s-coated apoptotic cells significantly enhanced macrophage production of TGF-beta1, expression of P-selectin, and secretion of IL-6. These data suggest that SM4s within tumors may promote apoptotic cell removal and alter the phenotype of tumor-associated macrophages.

Implications for invariant natural killer T cell ligands due to the restricted presence of isoglobotrihexosylceramide in mammals.

Development of invariant natural killer T (iNKT) cells requires the presentation of lipid ligand(s) by CD1d molecules in the thymus. The glycosphingolipid (GSL) isoglobotrihexosylceramide (iGb3) has been proposed as the natural iNKT cell-selecting ligand in the thymus and to be involved in peripheral activation of iNKT cells by dendritic cells (DCs). However, there is no direct biochemical evidence for the presence of iGb3 in mouse or human thymus or DCs. Using a highly sensitive HPLC assay, the only tissue where iGb3 could be detected in mouse was the dorsal root ganglion (DRG). iGb3 was not detected in other mouse or any human tissues analyzed, including thymus and DCs. Even in mutant mice that store isoglobo-series GSLs in the DRG, we were still unable to detect these GSLs in the thymus. iGb3 is therefore unlikely to be a physiologically relevant iNKT cell-selecting ligand in mouse and humans. A detailed study is now warranted to better understand the nature of iNKT cell-selecting ligand(s) in vivo.

Expression of the class A macrophage scavenger receptor on specific subpopulations of murine dendritic cells limits their endotoxin response.

Dendritic cells (DC) function at the interface of innate and acquired immunity and are uniquely sensitive to specific stimuli. Pattern recognition receptors (PRR) on these cells are critically important because of their ability to recognise and initiate responses to conserved microbial-associated molecular signatures. With the exception of Toll-like receptors (TLR), we know relatively little about the specific distribution of other PRR amongst populations of DC. Here, we describe the expression of the murine class A macrophage scavenger receptor (SR-A) and show that it is restricted to specific subpopulations of bone marrow-derived and splenic DC. Importantly, we demonstrate that the receptor significantly alters the response of DC to endotoxin. In contrast to the activities of other PRR that have so far been examined, uniquely SR-A limits the maturation response; SR-A-/- cells display enhanced CD40 expression and TNF-alpha production. We discuss the potential contributions of SR-A to DC biology in the context of the known multiple activities of this receptor.

Identification and characterization of murine SCARA5, a novel class A scavenger receptor that is expressed by populations of epithelial cells.

Epithelia are positioned at a critical interface to prevent invasion by microorganisms from the environment. Pattern recognition receptors are important components of innate immunity because of their ability to interact with specific microbe-associated structures and initiate immune responses. Several distinct groups of receptors have been recognized. One of these, the scavenger receptors, has been classified into at least eight separate classes. The class A scavenger receptors are characterized by the presence of a collagen-like domain and include macrophage scavenger receptor type A (SR-A1 I/II, SCARA1) and MARCO (SCARA2). These receptors are known to make important contributions to host defense. Here, we identify a novel murine scavenger receptor, SCARA5, which has a structure typical of this class. The cDNA encodes 491 amino acids, which predict a type II protein that contains C-terminal intracellular, transmembrane, extracellular spacer, collagenous, and N-terminal scavenger receptor cysteine rich domains. Expression in Chinese hamster ovary cells confirmed that the receptor assembles as a homotrimer and is expressed at the plasma membrane. SCARA5-transfected cells bound Escherichia coli and Staphylococcus aureus, but not zymosan, in a polyanionic-inhibitable manner. Unlike other class A scavenger receptors, the receptor was unable to endocytose acetylated or oxidized low density lipoprotein. Quantitative RT-PCR and in situ hybridization demonstrate SCARA5 has a tissue and cellular distribution unique among class A scavenger receptors. Because of the restriction of SCARA5 transcripts to populations of epithelial cells, we propose that this receptor may play important roles in the innate immune activities of these cells.

The class A macrophage scavenger receptor attenuates CXC chemokine production and the early infiltration of neutrophils in sterile peritonitis.

The macrophage scavenger receptor (SR-A) is a multifunctional receptor that is associated with several important pathological conditions, including atherosclerosis. In this study, we show, using a sterile peritonitis model, that it can regulate the inflammatory response. SR-A null mice display an increased initial granulocytic infiltration because of overproduction of the CXC chemokines, MIP-2 and keratinocyte-derived cytokine. This differential response is dependent upon particle internalization and can be mimicked by advanced glycation end product-BSA-conjugated latex beads. Thus SR-A is a nonactivating receptor, which is the first example of a pattern recognition receptor that serves to counter the activities of proinflammatory receptors and attenuates the production of specific chemokines to ensure an inflammatory response of the appropriate magnitude.

Macrophagic response to human mesenchymal stem cell and poly(epsilon-caprolactone) implantation in nonobese diabetic/severe combined immunodeficient mice.

Nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice are extensively used to assess in vivo potentials for human cellular differentiation, development, and neophysiology. They are not only deficient in T and B cells, but also exhibit macrophage dysfunction and an absence of circulating complement. However, the survival of engrafted human mesenchymal stem cells (hMSCs) is limited and minimal mature bone tissue develops from implanted hMSCs in this model. The aim of the present study was to investigate the response to such implants in NOD/SCID mice. To this end, hMSCs genetically marked with enhanced green fluorescent protein, a biodegradable polymer, poly(epsilon-caprolactone) (PCL), and a bioconstruct incorporating the enhanced green fluorescent protein-labeled hMSCs with PCL after culture together for 3 weeks in vitro, were implanted into NOD/SCID mice and followed for up to 10 weeks. Monocytes/macrophages appeared to be the major invading cell type in all the implants and remained in the materials regardless of whether or not hMSCs were present over the time periods studied. When the hMSCs were implanted without the PCL scaffold, host macrophage invasion was also observed with the majority of hMSCs being eliminated within 2 weeks. Multinuclear giant cells or foreign body giant cells were seen in the cases of PCL implantation. These cells slowly infiltrated into the central core of the materials over a 10-week period of implantation with neutrophils and mast cells also being observed. In conclusion, in NOD/SCID mice, monocytes/macrophages still effectively respond to the implantation of xenografts and biopolymers with functional migration, phagocytosis, adhesion, foreign body recognition and formation of multinuclear giant cells, or foreign body giant cells. Thus, these animals still retain a level of innate immune responsiveness to these implantations and in addition may provoke a physiological environment that is unsuitable for extensive intramembranous ossification by engrafted hMSCs.

Inhibition of alpha-glucosidases I and II increases the cell surface expression of functional class A macrophage scavenger receptor (SR-A) by extending its half-life.

The class A scavenger receptor (SR-A) is a multifunctional trimeric membrane glycoprotein involved in atherogenesis. The mature receptor can mediate the binding and internalization of a number of specific ligands, including modified low-density lipoprotein. We have investigated the effects of inhibiting N-glycan processing on SR-A expression, distribution, and activity in the murine macrophage cell line RAW264.7. We have found that SR-A normally interacts with calnexin in the endoplasmic reticulum and in its mature form carries complex N-glycans. The imino sugar, N-butyldeoxynojirimycin (NB-DNJ) is an inhibitor of the N-glycan processing enzymes alpha-glucosidases I and II. Following NB-DNJ treatment SR-A became Endo H-sensitive, consistent with inhibition of N-glycan processing. A dose-dependent increase in cell surface expression of SR-A was observed in response to NB-DNJ treatment. The receptor on inhibitor-treated cells was still functional because the increased surface expression resulted in a proportional enhancement in the endocytosis of the ligand, acetylated low-density lipoprotein. The expression of SR-A on NB-DNJ cultured cells was further enhanced by co-treatment with interferon-gamma. Quantitative reverse transcriptase-PCR analysis did not show a significant difference in the amount of SR-A mRNA in NB-DNJ-treated RAW264.7 cells. However, the half-life of SR-A protein was significantly increased. These data indicate the retention of glucosylated N-glycans does not result in gross misfolding and degradation of this receptor or prevent its transport to the cell surface. SR-A interacts with calnexin and when the association is prevented changes in the recycling kinetics and rate of turnover of the receptor result, leading to enhanced cell surface expression.