Use of expression constructs to dissect the functional domains of the CHS/beige protein: identification of multiple phenotypes

The Chediak-Higashi Syndrome (CHS) and the orthologous murine disorder beige are characterized at the cellular level by the presence of giant lysosomes. The CHS1/Beige protein is a 3787 amino acid protein of unknown function. To determine functional domains of the CHS1/Beige protein, we generated truncated constructs of the gene/protein. These truncated proteins were transiently expressed in Cos-7 or HeLa cells and their effect on membrane trafficking was examined. Beige is apparently a cytosolic protein, as are most transiently expressed truncated Beige constructs. Expression of the Beige construct FM (amino acids 1-2037) in wild-type cells led to enlarged lysosomes. Similarly, expression of a 5.5-kb region (amino acids 2035-3787) of the carboxyl terminal of Beige (22B) also resulted in enlarged lysosomes. Expression of FM solely affected lysosome size, whereas expression of 22B led to alterations in lysosome size, changes in the Golgi and eventually cell death. The two constructs could be used to further dissect phenotypes resulting from loss of the Beige protein. CHS or beigej fibroblasts show an absence of nuclear staining using a monoclonal antibody directed against phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5) P2]. Transformation of beige j fibroblasts with a YAC containing the full-length Beige gene resulted in the normalization of lysosome size and nuclear PtdIns(4,5)P2 staining. Expression of the carboxyl dominant negative construct 22B led to loss of nuclear PtdIns(4,5)P2 staining. Expression of the FM dominant negative clone did not alter nuclear PtdIns(4,5) P2 localization. These results suggest that the Beige protein interacts with at least two different partners and that the Beige protein affects cellular events, such as nuclear PtdIns(4,5)P2 localization, in addition to lysosome size.     

Localization of the locus responsible for Chediak-Higashi syndrome in cattle to bovine chromosome 28

Chediak-Higashi syndrome in Japanese black cattle is a hereditary disease with prolonged bleeding time and partial albinism. In the present study, we mapped the locus responsible for the disease (CHS) by linkage analysis using microsatellite genotypes of paternal half-sib pedigrees obtained from commercial herds. Analysis revealed significant linkage between the CHS locus and marker loci on the proximal end of bovine chromosome 28. The CHS locus was mapped on the region incorporating the microsatellite markers BMC6020, BM2892, and RM016 with recombination fraction 0 and lod score 4.9-11.2. We also assigned the bovine CHS1/LYST, the homologue of the gene responsible for human Chediak-Higashi syndrome, to bovine chromosome 28 using a bovine/murine somatic cell hybrid panel. These findings suggest that a mutation in the CHS1/LYST gene is likely to be responsible for Chediak-Higashi syndrome in Japanese black cattle.     

LYST Controls the Biogenesis of the Endosomal Compartment Required for Secretory Lysosome Function

Chediak–Higashi syndrome (CHS) is caused by mutations in the gene encoding LYST protein, the function of which remains poorly understood. Prominent features of CHS include defective secretory lysosome exocytosis and the presence of enlarged, lysosome‐like organelles in several cell types. In order to get further insight into the role of LYST in the biogenesis and exocytosis of cytotoxic granules, we analyzed cytotoxic T lymphocytes (CTLs) from patients with CHS. Using confocal microscopy and correlative light electron microscopy, we showed that the enlarged organelle in CTLs is a hybrid compartment that contains proteins components from recycling‐late endosomes and lysosomes. Enlargement of cytotoxic granules results from the progressive clustering and then fusion of normal‐sized endolysosomal organelles. At the immunological synapse (IS) in CHS CTLs, cytotoxic granules have limited motility and appear docked while nevertheless unable to degranulate. By increasing the expression of effectors of lytic granule exocytosis, such as Munc13‐4, Rab27a and Slp3, in CHS CTLs, we were able to restore the dynamics and the secretory ability of cytotoxic granules at the IS. Our results indicate that LYST is involved in the trafficking of the effectors involved in exocytosis required for the terminal maturation of perforin‐containing vesicles into secretory cytotoxic granules.      

The road to lysosome‐related organelles: Insights from Hermansky‐Pudlak syndrome and other rare diseases

Lysosome‐related organelles (LROs) comprise a diverse group of cell type‐specific, membrane‐bound subcellular organelles that derive at least in part from the endolysosomal system but that have unique contents, morphologies and functions to support specific physiological roles. They include: melanosomes that provide pigment to our eyes and skin; alpha and dense granules in platelets, and lytic granules in cytotoxic T cells and natural killer cells, which release effectors to regulate hemostasis and immunity; and distinct classes of lamellar bodies in lung epithelial cells and keratinocytes that support lung plasticity and skin lubrication. The formation, maturation and/or secretion of subsets of LROs are dysfunctional or entirely absent in a number of hereditary syndromic disorders, including in particular the Hermansky‐Pudlak syndromes. This review provides a comprehensive overview of LROs in humans and model organisms and presents our current understanding of how the products of genes that are defective in heritable diseases impact their formation, motility and ultimate secretion.     

The enlarged lysosomes in beige j cells result from decreased lysosome fission and not increased lysosome fusion

Chediak-Higashi syndrome is an autosomal recessive disorder that affects vesicle morphology. The Chs1/Lyst protein is a member of the BEige And CHediak family of proteins. The absence of Chs1/Lyst gives rise to enlarged lysosomes. Lysosome size is regulated by a balance between vesicle fusion and fission and can be reversibly altered by acidifying the cytoplasm using Acetate Ringer's or by incubating with the drug vacuolin-1. We took advantage of these procedures to determine rates of lysosome fusion and fission in the presence or absence of Chs1/Lyst. Here, we show by microscopy, flow cytometry and in vitro fusion that the absence of the Chs1/Lyst protein does not increase the rate of lysosome fusion. Rather, our data indicate that loss of this protein decreases the rate of lysosome fission. We further show that overexpression of the Chs1/Lyst protein gives rise to a faster rate of lysosome fission. These results indicate that Chs1/Lyst regulates lysosome size by affecting fission.     

Chediak–Higashi Syndrome: A Rare Disorder of Lysosomes and Lysosome Related Organelles

Chediak–Higashi Syndrome (CHS) is a rare autosomal recessive disorder characterized by severe immunologic defects including recurrent bacterial infections, impaired chemotaxis and abnormal natural killer (NK) cell function. Patients with this syndrome exhibit other symptoms such as an associated lymphoproliferative syndrome, bleeding tendencies, partial albinism and peripheral neuropathies. The classic diagnostic feature of CHS is the presence of huge lysosomes and cytoplasmic granules within cells. Similar defects are found in other mammals, the most well studied being the beige mouse and Aleutian mink. A positional cloning approach resulted in the identification of the Beige gene on chromosome 13 in mice and the CHS1/LYST gene on chromosome 1 in humans. The protein encoded by this gene is 3801 amino acids and is highly conserved throughout evolution. The identification of CHS1/Beige has defined a family of genes containing a common BEACH motif. The function of these proteins in vesicular trafficking remains unknown.     

Altered composition and secretion of lysosome-derived compartments in Dictyostelium AP-3 mutant cells

Genetic alteration of the adaptor protein (AP)-3 complex is responsible for the type 2 Hermansky–Pudlak syndrome, a lysosomal storage disease similar to the Chediak–Higashi syndrome (CHS). AP-3 presumably participates in the biogenesis of late endosomal compartments and may also be critical for the regulated secretion of lysosomes by specialized cells. Here, Dictyostelium discoideum cells defective for the μ 3 subunit of the AP-3 complex were used and their phenotype analyzed. In μ3 mutant cells, endosomal maturation and lysosome secretion were markedly slower than that in wild-type cells. This phenotype is similar to that reported previously in lvsB mutant cells where the ortholog of the LYST gene, involved in CHS, is mutated. Detailed analysis revealed however significant differences between these two isogenic mutant cells: in lvsB mutant cells, the primary defect is an inefficient biogenesis of otherwise normal secretory lysosomes, while in μ3 mutant cells, the biogenesis and also the composition and the fusion properties of secretory lysosomes are affected. These results suggest that in D. discoideum , AP-3 controls both the efficiency and the specificity of postlysosome maturation, which represent two critical elements in the control of lysosome secretion.     

Analysis of the lysosomal storage disease Chediak-Higashi syndrome

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disorder of human, mouse (beige) and other mammalian species. The same genetic defect was found to result in the disease in all species identified, permitting a positional cloning approach using the mouse model beige to identify the responsible gene. The CHS gene was cloned and mutations identified in affected species. This review discusses the clinical features of CHS contrasting features seen in similar syndromes. The possible functions of the protein encoded by the CHS/beige gene are discussed, along with the alterations in cellular physiology seen in mutant cells.     

Hermansky-Pudlak syndrome and related disorders of organelle formation

Hermansky-Pudlak syndrome (HPS) consists of a group of genetically heterogeneous disorders which share the clinical findings of oculocutaneous albinism, a platelet storage pool deficiency, and some degree of ceroid lipofuscinosis. Related diseases share some of these findings and may exhibit other symptoms and signs but the underlying defect in the entire group of disorders involves defective intracellular vesicle formation, transport or fusion. Two HPS-causing genes, HPS1 and ADTB3A, have been isolated but the function of only the latter has been determined. ADTB3A codes for the beta 3A subunit of adaptor complex-3, responsible for vesicle formation from the trans-Golgi network (TGN). The many HPS patients who do not have HPS1 or ADTB3A mutations have their disease because of mutations in other genes. Candidates for these HPS-causing genes include those responsible for mouse models of HPS or for the 'granule' group of eye color genes in Drosophila. Each gene responsible for a subset of HPS or a related disorder codes for a protein which almost certainly plays a pivotal role in vesicular trafficking, inextricably linking clinical and cell biological interests in this group of diseases.     

Secretory lysosome biogenesis in cytotoxic T lymphocytes from normal and Chediak Higashi syndrome patients

The lytic proteins mediating target cell killing are stored in the lysosomes of activated cytotoxic T lymphocytes (CTL) and are secreted upon recognition of a target cell. These secretory lysosomes cannot be detected in resting T lymphocytes. Interaction of a resting cell with a target cell activates de novo formation of secretory lysosomes. CTL clones in culture mimic this behaviour, and so provide an ideal system for studying secretory lysosome biogenesis and maturation. In the genetic disease, Chediak Higashi syndrome (CHS), all lysosomes in the cells are enlarged and reduced in number compared with wild-type (WT) cells. We have used CTL from this disease to study secretory lysosome biogenesis and maturation. We show that at early stages after activation the secretory lysosomes are identical in WT and mutant cells, and that delivery of proteins to the secretory lysosome along the biosynthetic and endocytic pathways is normal in the mutant cells. With time, the lysosomes in the mutant cells aggregate, become larger and fewer in number and eventually form giant structures. Our results show that the initial steps of secretory lysosome formation are normal in CHS, but that the organelles subsequently fuse together during cell maturation to form the giant secretory lysosomes.