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Prepublished online as a Blood First Edition Paper on November 21, 2002; DOI 10.1182/blood-2002-09-2789.
IMMUNOBIOLOGY
From the Unité de Recherche sur le
Développement Normal et Pathologique du Système Immunitaire
Institut National de la Santé et de la Recherche Médicale
(INSERM) U429, Hôpital Necker-Enfants Malades, Paris,
France; and équipe Motilité structurale and
équipe Mécanismes moléculaires du transport
intracellulaire, Unité Mixte de Recherche, Institut
Curie/Centre National de la Recherche Scientifique 144, Institut Curie,
Paris, France.
Rab27a is a member of the Rab family of small GTPase proteins, and
thus far is the first member to be associated with a human disease (ie, the Griscelli syndrome type 2). Mutations in the Rab27a gene cause pigment as well as cytotoxic granule transport defects, accounting for the partial albinism and severe immune disorder
characteristics of this syndrome. So far, 3 Rab27a missense mutations
have been identified. They open a unique opportunity to designate
critical structural and functional residues of Rab proteins. We show
here that the introduction of a proline residue in the The Rab family of low-molecular-weight GTPases
plays an essential role in intracellular transport vesicles by
controlling membrane trafficking between intracellular compartments.
Rab GTPases interact specifically with a subset of effector proteins
that, in turn could impart specificity to membrane trafficking at the levels of vesicle translocation, docking, and fusion.1-3
In mammalian cells, more than 50 Rab proteins have now been
identified.4-6 Rab27a is the only one so far that has been
associated with a human disease (ie, the Griscelli syndrome). We
previously reported that the Griscelli syndrome, characterized by a
partial albinism of the skin and hair and a severe immunologic
disorder, results from Rab27a deleterious
mutations.7 Although Rab27a is wildly expressed, the
clinical phenotype of the patients with Griscelli syndrome indicates a
determinant role of this Rab protein in melanocytes and lymphocytes. In
addition to its role in melanosome peripheral transfer, we have shown
that Rab27a is required for cytotoxic granule exocytosis, and that the
defective cytotoxic activity of patients' lymphocytes resulting from
Rab27a mutation accounts for the severe immune disorder characteristic
of this syndrome, known as hemophagocytic syndrome
(HS).7,8 HS is characterized by lymphoid organ and
extranodal infiltration by polyclonal activated T cells, mostly of the
CD8+ subset, and by activated macrophages that phagocytose
blood cells. This condition does not, in most cases, spontaneously
remit. Immune cell infiltration results in massive tissue necrosis,
organ failure, and death in the absence of immunosuppressive treatment.
Massive cytokine release is another hallmark of the condition, and it is thought that macrophage activation is mostly the consequence of
tumor necrosis factor (TNF) production, leading to
hemophagocytosis, high triglyceride levels, and coagulation disorders.
Similarly, in the ashen mice,
Rab27a mutation leads to defects in melanosome transport and
granule exocytosis in cytotoxic T
lymphocytes.9-11 Recently, the product of the
gene mutated in the leaden (Mlphd)
mice,12 the melanophilin (Mlph; also known as Slac2-a),
was identified as an effector of Rab27a in
melanocytes.12-15 The Mlph bridges Rab27a and the
molecular motor myosin Va, the tripartite protein complex being
required for melanosome transport function.16,17
The molecular mechanisms, which underlie the diversity of Rab/effector
interaction, are not yet fully understood. Rabs cycle between a
guanosine triphosphate (GTP)-bound (active) and a guanosine diphosphate
(GDP)-bound (inactive) conformation, but this highly conserved
mechanism among GTPase proteins cannot account for the discrimination
of diverse effectors and regulatory factors. In addition to
Rab-specific motifs present in all Rab GTPase, Rab subfamily-specific
sequences (RabsF), with high-amino-acid conservation within
subfamilies, have been identified and are thought to participate in the
specificity of regulator interaction.5 Crystallographic studies of the constitutively active mutant of Rab3A and its effector, rabphilin3A, have defined 3 hypervariable Rab complementary-determining regions (Rab CDRs) involved in molecular interaction. They appear as
major determinants of functional effector specificity.18 Interestingly, all 3 Rab CDRs are included in the subfamily motifs defined by sequence homology analysis.5 An invariant
hydrophobic triad at the switch interface of active Rab3A was shown to
mediate central interaction with rabphilin3A.18 Spatial
exposition of this triad in the active form of Rab5C is
noncomplementary to the switch interaction epitope of rabphilin3A,
supporting the specific contribution of these hydrophobic residues in
effector recognition.19 Rab3A and Rab27a are
phylogenetically similar and share 43% identity of amino acid
residues. Several effectors of Rab27a have been described either
belonging to the synaptotagmin-like protein (Slp) family (Slp1/JFC1,
Slp2, Slp3, Slp4/granuphilin, and Slp5) or to the Slac2 (Slp homologues
lacking C2 domain) family (Slac2/melanophilin, Slac2b, and
Slac2c/MyRIP). All of them were shown to interact with Rab27a through a
conserved N-terminal Slp homology domain (SHD), which was identified by
Kuroda et al.14 The Rab effector domain of
rabphilin-3A and the SHD domain of melanophilin are conserved and share
26% identity and 43% similarity. Therefore, a closely similar
structural interaction is likely to occur as confirmed by the ability
of rabphilin-3A to specifically interact in vitro with
Rab27a.14
Most Rab27a mutations so far identified in Griscelli syndrome patients
are null mutations resulting from nonsense mutations or frameshift
deletions (Ménasché et al7; and G.d.S.B.,
personal data, October 2002). However, we here identified 3 inherited homozygous missense mutations in the Rab27a gene
(Trp73Gly, Leu130Pro, and Ala152Pro) that are associated with Griscelli
syndrome.7 These spontaneous mutants represent a
unique opportunity to evaluate how these designated critical residues
of Rab27a, and more broadly, perhaps, of Rab proteins, may participate
in Rab protein functions. We thus analyzed nucleotide-binding activity
and Mlph-binding ability, as well as the potential transdominant effect
of these various mutants expressed in melanocytes and cytotoxic cells. We report that the insertion of a proline residue either in the Patients
Molecular cloning of human Rab27a cDNA and mutants
Wild-type (WT) Rab27a cloning.
A cDNA encoding a full open-reading frame of Rab27a was obtained by
reverse transcription-polymerase chain reaction (RT-PCR), using SuperScript One-Step RT-PCR System with platinum Taq (Invitrogen, Cergy Pontoise, France) from total RNA of human B-EBV lymphoblastoid cell line as described previously. The following primers were used:
EcoRI-Rab27a-Met-sense,
5'-GGAATTCCATGTCTGATGGAGATTATGA-3' and
BamHI-Rab27a-stop-antisense,
5'-GCGGATCCGCCCACCTGAACTACTATGTCA-3'. Sequence site
recognition of the corresponding enzymes are underlined. Digested PCR
product was cloned into pcDNA 3.1 (Invitrogen), pFlag-CMV-4 (Sigma,
Saint Quentin Fallavier, France), and pEGFP-C1 (Clontech, Basingstoke,
United Kingdom). For in-frame cloning with His6-tag in
P15b,22 the following primers were used to generate the
PCR product: SmaI-Rab27a-Met,
5'-TCCCCCGGGGATATGTCTGATGGAGATTATGA-3' sense primer; and
BamHI-Rab27a-stop antisense primer.
Mutants Rab27a cloning.
Site-directed mutagenesis (in bold on the sequences) of
Rab27a was performed using the double-PCR strategy.23 The
primers used are as follows: 5'-TGTAGGGAAGAACAGTGT-3'
(Thr23Asn primer; sense); 5'-TGGTAAAGTACACTGTTCTT-3'
(Thr23Asn primer; antisense); 5'-GCAGTTAGGGGACAC-3'
(Trp73Gly primer; sense); 5'-GTCCCCTAACTGCAGGTGGATT-3'
(Trp73Gly primer; antisense); 5'-GCAGTTACTGGACAC-3'
(Trp73Leu primer; sense); 5'-GTCCAGTAACTGCAGGTGGATT-3' (Trp73Leu primer; antisense); 5'-GCAGTTATCGGACAC-3'
(Trp73Ser primer; sense); 5'-GTCCGATAACTGCAGGTGGATT-3'
(Trp73Ser primer; antisense); 5'-GGCTGGAGAGGTTTCGT-3'
(Gln78Leu primer; sense); 5'-CTCCAGCCCTGCTGTG-3' (Gln78Leu
primer; antisense); 5'-AGTGCCGTGTGGAAACAA-3' (Leu130Pro
primer; sense); 5'-TTGTTTCCACACGGCACT-3' (Leu130Pro primer;
antisense); 5'-ATAGCACTCCCAGAGAAA-3' (Ala152Pro primer;
sense); and 5'-TTTCTCTGGGAGTGCTAT-3' (Ala152Pro primer;
antisense). The amplified PCR products were digested by SmaI
and BamHI and subcloned into pcDNA 3.1 Pet15b/His and
pFlag-CMV-4 and pEGFP-C1 vectors.
Molecular cloning of the SHD domain of human Mlph
Purification of recombinant proteins Escherichia coli BL21 cells were transformed with recombinant plasmid Pet15b containing the correct inserts and grown in Luria-Bertani (LB) medium with 30 µg/mL ampicilin. When optical density (OD600) was 0.6, isopropyl-1-thio- -D
galactopyranoside was added to a final concentration of 1 mM.
Cells were harvested 3 hours later by centrifugation at
1500g for 10 minutes. Bacterial pellets were resuspended in
10 mM Tris-HCl, pH 8.8; 300 mM NaCl; 1 mM dithiothreitol (DTT); 10 mM
imidazol; 10 µM GTP; and 10 µg/mL phenylmethylsulfonyl fluoride
(PMSF), and sonicated at 4°C. The lysate was centrifuged twice at 10 000g for 10 minutes. The mutants Leu130Pro and
Ala152Pro were solubilized in 0.8% sodium dodecyl sulfate
(SDS), 250 µM triethanolamine, 1.5% sarcosyl, and 0.5 M EDTA
(ethylenediaminetetraacetic acid). Purification was carried
out according to the instruction manual of the
QIAexpressionist (Qiagen SA, Courtaboeuf, France). Briefly,
the supernatant was incubated with 300 µL nickel-nitrilo tri-acetic
acid (Ni-NTA) resin and mixed gently for 2 hours at 4°C. The
recombinant proteins were washed and eluted with 250 mM imidazol.
Before the binding assays, the proteins were dialyzed overnight against
20 mM Tris-HCl, pH 7.5; 1 mM DTT; and 5 mM MgCl2. The
purity was estimated by SDS-polyacrylamide gel electrophoresis (PAGE)
and coomassie blue staining. Protein concentration was determined by a Bradford assay (Pierce-Perbio Science, Bezons, France)
using bovine serum albumin (Sigma) as a standard.
Binding of nucleotides Recombinant proteins were incubated in 20 mM Tris-HCl, pH 7.5; 1 mM DTT; 5 mM MgCl2; 10 mM EDTA; 0.5 g/L bovine serum albumin; (3H)GTP or (3H)GDP (7.7 Ci/mmol [284.9 GBq/mmol], Amersham Pharmacia Biotech, Saclay, France); and cold 30 µM GTP or GDP. After incubation at 30°C for the indicated times, samples were diluted in 500 µL ice-cold washing buffer (20 mM Tris-HCl, pH 7.5; 25 mM MgCl2; and 100 mM NaCl) and applied to a nitrocellulose filter (0.45 µm; Millipore, Saint Quentin en Yvelines, France). The filters were rinsed with 4 × 4 mL ice-cold washing buffer, and the radioactivity retained on the filters was determined by scintillation counting.24GTPase activity In the condition described above, proteins were preincubated for 1 hour for nucleotide binding with ( -32P) GTP (5000 Ci/mmol [185 000
GBq/mmol], Amersham Pharmacia Biotech), and then 20 mM
MgCl2 was added to the final concentration. At the
indicated times, samples were diluted with cold buffer, filtered, and counted.
Cell culture, transient transfection, and functional assay The immortal control-mouse melanocyte cell line, melan-a, was kindly provided by Dorothy Bennett, (St George's Hospital Medical School, London, United Kingdom). Melan-a was cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and 200 nM phorbol myristate acetate (PMA; Sigma) at 37°C with 10% CO2. For microscopic analysis, cells were grown on coverslips for 24 hours and cotransfected using the liposomal transfection reagent Fugene 6 (Roche Diagnosis, Meylan, France) with 2.5 µg of pEGFP-C1 (Clontech), allowing soluble green fluorescence protein (GFP) expression, and 2.5 µg of pcDNA containing Rab27a WT or mutants and/or Mlph. Cells were fixed 24 hours later in 3.7% paraformaldehyde for 15 minutes, washed extensively, and mounted in a medium containing Mowiol antifading agent (Calbiochem, San Diego, CA). Cells were observed using an Axioplan 2 microscope (Zeiss, Jena, Germany). The 293T cell line25 was cultured in Dulbecco modified Eagle medium (DMEM) with 10% FCS at 37°C with 5% CO2 and transfected by electroporation (250 V, 900 µF [EquiBio, Ashford, United Kingdom]). Herpesvirus saimiri-transformed CD8+ T cells26 were kept continuously in medium containing interleukin-2 (IL-2; 40 U/mL). This T-cell line was transiently transfected with various EGFP-Rab27a constructs using the Human T-cell Nucleofector Kit (Amaxa Biosytem, Köln, Germany), and degranulation assay was tested 18 hours later using N-a-benzyloxycarbonyl-L-lysine thiogbenzyl ester (BLT) esterase release analysis. Intracellular expression of the various constructs was evaluated by FACScan analysis (Becton Dickinson, San Diego, CA). Peripheral blood lymphocytes from patients or controls were stimulated with phytohemagglutinin (PHA, 1:700 dilution; Difco, Detroit, MI) for 24 hours and then cultured in IL-2 (40 U/mL; Valbiotech, Paris, France) for 6 days. The degranulation assay was done on these cells as previously described,7 with some modifications. Briefly, anti-CD3 antibody (20 µg/mL; OKT3, Ortoclone; Jansen, France) coated in 96-well plates was used instead of target cells to stimulate 6 × 105 T cells/well or 2.5 × 105 transfected H saimiri CD8+ T cells/well in a 4-hour release assay. The release of BLT esterase activity on a 50-µL aliquot of cell-free supernatant was performed as previously described.7Immunoprecipitation Flag-Rab27a WT or mutants and 1-146Mlph-Myc were coexpressed in 293T cells. One day after electroporation, cells were lysed in 50 mM Tris, pH 8.0; 1% nonidet P-40; and 2 mM EDTA, supplemented with 10 µg/mL each of protease inhibitors leupeptin, aprotinin, N-tosyl-L-phenylalanine chloromethyl ketone, N-p-tosyl-L-lysine chloromethyl ketone, and PMSF, as well as the phosphatase inhibitors sodium fluoride (50 mM) and sodium orthovanadate (1 mM).27 Anti-c-Myc (9E10; Santa Cruz Biotechnology, Hedelberg, Germany) and anti-Flag-M2 (Sigma) were used for immunoprecipitation. Immune complexes were collected with protein G (Sigma). Proteins were loaded in 12% SDS-PAGE and transferred onto Immobilon membrane (Millipore).
Nucleotide binding and GTPase activity of Rab27a mutants There were 3 different homozygous missense mutations of the Rab27a gene found to be associated with Griscelli syndrome (Leu130Pro, Ala152Pro, and Trp73Gly), pointing out the biologic relevance of these amino acid transitions. Based on the crystal structure of GTP-Rab3A,18 Leu130Pro is located on the5 sheet, Ala152Pro on the  4 loop, and Trp73Gly on the 3
sheet near the nucleotide-binding site (Figure
1). To evaluate the molecular
consequences of these substitutions, the Rab27a mutants were expressed
and tested for their nucleotide-binding ability as well as GTPase
activity. In addition, the constitutively GTP-bound, active Rab27a
Gln78Leu mutant (equivalent to Rab3A Gln81Leu in Figure 1), as well as the Rab27a Thr23Asn mutant (corresponding to Rab3A Thr36Asn in Figure
1), which is predominantly in the GDP-bound, inactive conformation, were also constructed and tested. As expected, WT Rab27a and Gln78Leu mutant bind to GTP (Figure 2A). Mutant
Trp73Gly was shown to exhibit a similar GTP-binding ability, although
with lower kinetics. These 3 proteins also bind to GDP as does the
Thr23Asn mutant (Figure 2B). In contrast, Leu130Pro and
Ala152Pro mutants did not bind to GTP or GDP nucleotides (Figure 2A-B).
The introduction of a proline at the mutation sites most likely impairs
the folding of these proteins as suggested by the specific detergent
conditions required for their purification. Since both GTP- and
GDP-binding activity were maintained when WT was prepared in the same
conditions (data not shown), defective binding activity appears
strictly related to the presence of the proline and not to the
experimental procedure used.
The GTPase activity of the Rab27a proteins was next evaluated. Only the
WT form of Rab27a had an intrinsic GTP hydrolysis activity, whereas
Trp73Gly and Gln78Leu were inefficient in hydrolyzing GTP (Figure 2C).
By analogy to other small GTPase proteins, the conserved Gln78 residue
is expected to stabilize the catalytic transition state between switch
I and II. Transition to leucine may lower this catalytic rate and lock
the Rab protein into the activated, GTP-bound state. Residue 73 (residue 76 of Rab3A in Figure 1) is located in the Mutations at the Trp73 residue of Rab27a, a component of the invariant hydrophobic triad, impair effector binding A most interesting observation was that the spontaneous mutant Trp73Gly, which was observed in 2 patients with Griscelli syndrome, precisely affected one residue of the invariant hydrophobic triad suspected of imposing critical conformational constraints on the active form of the Rab protein, thus increasing effector-binding specificity. We have thus evaluated the ability of this mutant to interact with one of its recently described effectors, melanophilin. The specific interaction of the SHD domain of Mlph, with WT Rab27a or its mutants, was investigated in vivo using a Myc-tagged SHD domain of Mlph (Myc-SHD) and FLAG-tagged Rabs, which were coexpressed in 293T cells. As expected, Myc-SHD protein was specifically found coimmunoprecipitated with the FLAG-Rab27a protein either in its wild-type form or Gln78Leu form, whereas the inactive GDP-bound form Thr23Asn was not (Figure 3A). Although the Trp73Gly mutant of Rab27a exhibits both the same GTP-binding activity and intrinsic GTPase deficiency as the constitutively active form Gln78Leu, the association of Trp73Gly construct with Myc-SHD could not be detected (Figure 3B). Similarly, 2 other mutants at position 73 (Trp73Leu and Trp73Ser) shown to efficiently bind GTP (Figure 1D) were unable to associate with the SHD domain of Mlph (Figure 3B). The same results were observed by immunoprecipitation with anti-Flag antibody or anti-Myc antibody (data not shown).
These results highlight the specific requirement of the conserved residue at position 73 for melanophilin binding, even with a GTP-bound state of the Rab27a protein. These data strongly support the contribution of a proper positioning of the invariant hydrophobic triad in effector interaction. Overexpression of Rab27a Trp73 mutants does not modify melanosome distribution To address the function of the Trp73 Rab27a mutants, the effect of their overexpression on melanosome distribution in melanocytes was analyzed. Transient overexpression of Trp73Gly in melan-a, a wild-type mouse melanocyte cell line, did not significantly affect the localization of the pigment granules, which were evenly distributed throughout the cytoplasm (Figure 4C-D) as observed in control enhanced GFP (EGFP)-transfected cells (Figure 4A-B). Similarly, Trp73Leu and Trp73Ser overexpression as well as Thr23Asn mutant overexpression in melan-a did not significantly modify melanososome distribution (not shown). In contrast, overexpression of the constitutively active Gln78Leu Rab27a mutant resulted in dramatic redistribution of pigment granules, which, in more than 50% of the transfected cells, were clustered in the perinuclear region (Figure 4E-F). A dominant-negative effect was also mediated by the overexpression in this cell line of the SHD domain of the Mlph able to interact with Rab27a (Figure 4G-H). Mlph truncated from its C terminal part cannot target myosin Va to melanosome, thus affecting melanosome mobility. Similar effects on granule positioning were observed for these various mutants used either as nontagged protein (as shown in Figure 4), whose expression was assessed by Western blot (data not shown), or tagged with an EGFP or Flag protein (not shown). Intracellular localization analysis of the transfected constructs fused with EGFP showed that the mutants' constructs were mostly detected in the cytosol rather than associated with the membrane fraction (not shown). Together, these results suggest that in these cells, because of the inability of Trp73 mutants as well as Thr23Asn mutant to bind to endogenous Rab27a effector(s), these mutants do not interfere further with the endogenous Rab27a function. In contrast, the redistribution of melanosomes observed in Rab27a Gln78Leu-transfected cells may result from the rerouting and stable binding of the total pool of endogenous Rab27a effector(s) by this active mutant highly expressed in the cytosol. In this situation, the endogenous Rab27a protein is likely deprived of its effector(s) association, essential for the capture and peripheral movement of melanosomes. However, a dominant-negative effect of Rab27a Gln78Leu mutants has not been observed in previously reported studies.28,29 To better analyze the mechanism of the dominant-negative effect observed with the Gln78Leu mutant construct in our study, we investigated whether a normal phenotype could be rescued in melan-a cells cotransfected with both the SHD domain of Mlph and the Rab27a Gln78Leu mutant. The normal distribution of melanosomes observed in this situation (Figure 4I-J) strongly supports our hypothesis of a dominant-negative effect of Gln78Leu through endogenous effector capture. In addition, the absence of Trp73 mutants' effect in the same situation indirectly demonstrates in vivo the requirement of the Trp73 residue of Rab27a in specific melanophilin-effector binding.
Overexpression of Gln78Leu, but not of Trp73Gly inhibits cytotoxic granule exocytosis We have previously shown that Rab27a mutations in patients with Griscelli syndrome affect cytotoxic granule exocytosis, a finding further confirmed in the ashen murine model.10,11 Granule exocytosis was measured by analyzing the release into the supernatant of the BLT esterase (granzyme A) activity following lymphocyte TCR triggering; this was compared with the total BLT-esterase activity contained in the lymphocyte granules.7 Granule exocytosis from patients' lymphocytes with Trp73Gly transition was undetectable, whereas in the same conditions, 50% of the granule content of control lymphocytes was released (Figure 5A). A dominant effect associated with overexpression of the Rab27a mutants was similarly assessed on granule content release from an H saimiri-transformed cytotoxic CD8+ T-cell line. Transient overexpression of EGFP/Rab27a-Trp73Gly construct did not significantly modify enzyme release when compared with wild-type construct effect (Figure 5B). In contrast, overexpression of the constitutively active mutant Gln78Leu resulted in a significant inhibition of cytotoxic granule exocytosis (Figure 5B). Thus, as observed in melanocytes overexpressing the mutant constructs, Gln78Leu, but not the Trp73Gly construct, interferes with the endogenous Rab27a intracellular pathway in lymphocytes. These results strongly suggest that in both cell types, Rab27a-specific effectors' binding requires a spatial conformation of the triad, which is affected when Trp73 is mutated.
We herein report the molecular and functional analysis of 3 missense mutations in the Rab27a gene previously identified as resulting in the expression of Griscelli syndrome
phenotype.7 The introduction of a proline in the Rab27a
protein, located either in the Functional difference between these Trp73 and Gln78 mutants characteristics was also evidenced by their distinct effect on melanosome relocalization in melan-a cells, as well as in cytotoxic granule release. Gln78Leu mutant was able to exert only a dominant-negative effect, leading to perinuclear clustering of melanososomes in melanocytes and inhibition of cytotoxic granule exocytosis in lymphocytes. This dominant-negative effect resulted from the sequestration of an upstream activator, since it was inhibited by the coexpression of melanophilin in melanocytes. In accordance with this observation, all other mutants, including Trp73Gly, unable to coimmunoprecipitate with melanophilin did not modify melanosome transport. It is worth noting that a dominant-negative effect of the inactive Thr23Asn mutant has been reported (in 13%28 to 80%29 of the melanocytes), whereas the active Gln78Leu construct had no effect. In our study, mutants have the same effect in melanocytes and in cytotoxic cells; this is true with either tagged or untagged expressed Rab27a constructs of these mutants, thus strengthening the specificity of the observed effects. Such variations may result from differences in Rab27a mutants' expression level and/or in differential membrane association of the Rab27a mutants' constructs among experiments. Although Mlph was shown to be expressed in melanocytes, its presence is undetectable in cytotoxic cells, and accordingly, mutation in Mlph, which characterized leaden mice, does not affect cytotoxic activity.16 It was thus hypothesized either that another member of the Mlph family specifically interacts with Rab27a for inducing cytotoxic granule exocytosis or that structurally unrelated effectors regulate this function in cytotoxic cells. The observation that Gln78Leu but not Trp73Gly exerts a dominant-negative effect on cytotoxic granule exocytosis is in favor of the former hypothesis, since interaction of Rab27a with a specific effector in cytotoxic cells requires fine structural constraints indistinguishable from the one enabling Rab27a-Mlph interaction in melanocytes. Future experiments should thus identify the specific effector(s) of Rab27a in cytotoxic cells, a functionally critical molecule in triggering cell cytotoxicity. All the patients with Griscelli syndrome due to Rab27a mutation developed an HS consecutive to the functional impairment of the secretory cytotoxic pathway. Failure to kill antigen-presenting cells by effector cytotoxic cells, can favor a sustained immune response, and therefore the persistence of overwhelming activated T lymphocytes. These T lymphocytes will lead to macrophage activation, secretion of inflammatory cytokines and their subsequent deleterious effect in infiltrated organs.8 Although comparison of disease severity among patients from various geographic and medical contexts is a difficult task, it is interesting to note that patients with either Ala152Pro or Trp73Gly missense mutations had a later onset of HS than patients with null mutation. This suggests a possible in vivo residual activity of these mutated Rab proteins. Further analysis of mutated Rab27a proteins could contribute to both a better assessment of the structure/function relationship of Rab27a and of in vivo immunologic comparison.
We thank Dorothy Bennett, Helmut Fickenscher, and Pier Giorgio Natali for providing us with melan-a cells, H saimiri C3 T cells, and MNT1 cell line, respectively.
Submitted September 13, 2002; accepted November 12, 2002.
Prepublished online as Blood First Edition Paper, November 21, 2002; DOI 10.1182/blood-2002-09-2789.
Supported by grants from INSERM, association de Recherche sur le Cancer (ARC 5849; G.de S.B. and B.G.), and association Vaincre les Maladies Lysosomales (VML).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Geneviève de Saint Basile; INSERM U 429, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, Paris Cedex 15, 75743 France; e-mail: sbasile{at}necker.fr.
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Top
Abstract
Introduction
Griscelli syndrome type 2 (G.d.S.B.,
personal data, October 2002). Mutations in 3 of these patients
(P9, P15, and P17) have been previously reported.
), Trp73Gly (gray triangle), and Gln78Leu
(inverted gray triangle). The WT and mutant Rab27a proteins were loaded
with (
Gly
transition may have increased switch flexibility and thus impaired the
dynamics of the conformational changes associated with each nucleotide binding. To test this hypothesis, 2 other mutations at position 73 were
generated: either a leucine residue (Trp73Leu), as in the p21 Ras
protein, or a serine residue (Trp73Ser), which is supposed to limit
switch II conformational liability possibly introduced by the
glycine residue. Although these mutants could bind GTP and GDP
with high affinity (Figure 
). Transfection efficiency for each
construct was assessed by FACScan analysis of EGFP expression (
).
The results shown are representative of 2 independent experiments with
similar results.