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The range of possible mutations in Btk that result in X-linked agammaglobulinemia is unknown. Reported mutations in Btk include point mutations predicted to halt Btk activity and mutations that result in the deficient expression of Btk8,9,11. In this report we describe a single point mutation in the SH2 domain of Btk in a B-cell line from a patient with atypical X-linked agammaglobulinemia. SH2 domains are critical mediators of binding with phosphotyrosine-containing proteins in the cell12. The mutation is located in what crystal-structure studies of the src SH2 domain predict is a critical hydrophobic binding pocket13. The consequence of this mutation is decreased stability of the Btk protein, possibly resulting from the inability of Btk to interact with important substrates. This mutation and other spontaneous mutations in Btk should help further clarify the function of Btk in normal B-cell development.
Methods
Patient's Characteristics
The patient was a 23-year-old man who was the oldest of three brothers previously described as having atypical X-linked agammaglobulinemia14. All three brothers were given a diagnosis of hypogammaglobulinemia in 1976 when the proband was six years old and his brothers were five and two years old. Before the initiation of intramuscular gamma globulin therapy, the patient's serum IgG concentration was 590 mg per deciliter, his IgM concentration was 18 mg per deciliter, and his IgA concentration was below 5 mg per deciliter. His five-year-old brother had an IgG concentration of 140 mg per deciliter, and IgM and IgA concentrations of less than 5 mg per deciliter. The youngest had an IgG concentration of 470 mg per deciliter, and IgM and IgA concentrations of less than 5 mg per deciliter. All three brothers had 0.3 to 2 percent B cells in the peripheral circulation, whereas patients with typical X-linked agammaglobulinemia have a mean of 0.1 percent and normal subjects have 5 to 15 percent B cells15. Although the patient poorly complied with therapy, he had not had serious infections.
Cell Lines
B-cell lines transformed by Epstein-Barr virus (EBV) were derived from the patient's peripheral-blood lymphocytes infected with supernatants from the marmoset cell line B95-816. A control cell line was derived in a similar manner from an obligate female carrier of X-linked agammaglobulinemia and was kindly provided by Max Cooper (University of Alabama, Birmingham).
RNA Analysis
RNA was extracted from EBV-transformed B-cell lines with RNAzol (Cinna/Biotecx), and 20 µg of total RNA was subjected to electrophoresis on a formaldehyde-agarose gel, transferred to nitrocellulose filters, and probed with 32P-labeled human Btk cDNA. The Btk and 
-actin complementary DNA (cDNA) probes were used as previously described8.
Protein Analysis
For metabolic labeling 1 x 107 EBV-transformed B cells were incubated for four hours with 250 micro Ci of 35S-labeled methionine per milliliter in methionine-free medium and immunoprecipitated with anti-Btk antibody as described previously8. In vitro kinase assays were performed without sodium dodecyl sulfate and in the presence of 20 mM manganese as described previously8. For Western blotting, protein concentrations from cell lysates were determined with a Bio-Rad detection kit. A total of 25 µg of protein was suspended in sample buffer with 2-mercaptoethanol, subjected to electrophoresis on a 10 percent polyacrylamide gel, and electrophoretically transferred to nitrocellulose filters. The filters were incubated for two hours at room temperature with anti-Btk antibody, washed, and then incubated for one hour with horseradish peroxidase-conjugated goat antirabbit antibody (Bio-Rad). The filters were developed with an enhanced chemiluminescence kit (Amersham) and exposed to film.
Amplification and Sequencing
RNA was prepared from EBV-transformed B cells with guanidium thiocyanate17. RNA was reverse transcribed to cDNA and amplified by the polymerase chain reaction (PCR) with specific primer pairs derived from the published sequence for human Btk cDNA9. Amplification consisted of denaturation at 94 °C for 5 minutes followed by 30 cycles of denaturation at 94 °C for 1 minute, annealing at 58 °C for 1 minute, and extension at 72 °C for 1.5 minutes. Overlapping fragments were cloned into a TA vector kit (Invitrogen) and sequenced from universal M13 primers or oligomers from human Btk cDNA.
Results
The expression of the Btk protein was analyzed at the messenger-RNA (mRNA) and protein levels in a B-cell line derived from the patient with atypical X-linked agammaglobulinemia14 and from a control subject who was an obligate carrier of X-linked agammaglobulinemia. Full-sized mRNA was expressed at normal levels as compared with levels in the control (Figure 1A). Metabolic pulse-labeling with 35S-labeled methionine revealed that the amount of Btk protein synthesized by the patient's cells was also normal as compared with that of the control (Figure 1B). However, steady-state levels of Btk protein, as determined by Western blot analysis, were lower (Figure 1C). Similarly, protein kinase activity, as measured by an in vitro autophosphorylation assay, was also reduced as compared with that of the control, and was significantly lower than predicted by pulse-labeling (Figure 1D). Thus, although the rate of protein synthesis as determined by metabolic pulse-labeling was not affected, the level of total protein expressed was low and correlated with reduced kinase activity. Taken together, the results indicate a problem of stability rather than catalytic activity, as has been described for other mutations of the Btk gene9.
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We describe a novel mutation in the gene encoding Btk in atypical X-linked agammaglobulinemia. This mutation, in the SH2 domain, allows the expression of normal levels of Btk transcript but encodes a protein with decreased stability. This observation supports the hypothesis that atypical X-linked agammaglobulinemia is due to more subtle mutations in the gene responsible for typical X-linked agammaglobulinemia14. Btk is likely to function by means of multiple intracellular signaling pathways. Subtle mutations may block signaling in one pathway while alternative pathways continue to function. This may be analogous to another less severe mutation in the Btk gene in the X-linked immunodeficient (Xid) mouse strain19,20. This defect is characterized by a lack of phenotypically and functionally diverse B cells, a lack of T-independent type II antibody responses, and abnormal responsiveness to activation signals21. Although the mutation may alter intracellular interactions, the expression of Btk and autokinase activity are normal.
Cytoplasmic tyrosine kinases like Btk act as intermediaries in signal transduction after they are activated from the cell surface or from within the cell10. Activation results in a cascade of events, including phosphorylation of proteins that regulate the interaction with components downstream of the pathway. The SH2 domains of cytoplasmic tyrosine kinases are noncatalytic domains that span 100 amino acids and mediate the interaction with specific phosphotyrosine-containing molecules in the signaling pathway12. The decrease in protein stability resulting from the mutation in the SH2 domain of Btk in this patient may be explained by at least two mechanisms. One possibility is that this mutation leads to a conformational change in Btk that makes the protein unstable. Mutations in the SH2 domain of c-src have previously been shown to increase, decrease, or have no effect on the stability of c-src, with varied effects on the oncogenic potential of this molecule in vitro22. Decreased protein stability and decreased transforming activity have also been reported in activated forms of lck after the deletion of the SH2 domain23.
A second possible explanation is that the Btk SH2 domain binds to specific tyrosine-phosphorylated substrates that are important for stability or function and that are disrupted by the mutation identified in this patient. SH2 domains of cytoplasmic tyrosine kinases bind to tyrosine-phosphorylated proteins with high affinity13,24. Crystal-structure studies of the SH2 domains in src have defined a pocket that allows binding to phosphotyrosine-containing proteins13. Binding to the phosphotyrosine residue is mediated by a -pleated sheet, intervening loops, and an alpha helix. A second alpha helix forms the base of a hydrophobic binding site for three residues immediately following the phosphotyrosine-containing residue and may confer binding specificity13. The mutation in Btk in this patient was at amino acid residue 361, which on the basis of homology with the src SH2 domain is in the second alpha helix. This residue is totally conserved in the Btk gene family, and in src the corresponding residue is adjacent to a site that participates in hydrophobic peptide binding13,18. A mutation at this site in Btk may inhibit binding to critical tyrosine-phosphorylated substrates. Binding studies with mutated Btk SH2 domains may allow the identification of molecules that interact with Btk.
Supported in part by a grant (AI 12925) from the National Institutes of Health (NIH), a core grant (P30 CA21765) from the National Cancer Institute, funds from the American Lebanese and Syrian Associated Charities, and funds from the Federal Express Chair of Excellence (to Dr. Conley); by a grant (R35 CA53867) from the NIH and the Howard Hughes Medical Institute (to Dr. Witte); by funds from the Leukemia Society of America (to Dr. Saffran); by an NIH Immunology Training Grant, funds from the Jonsson Comprehensive Cancer Center, and a grant (AR36834) from the NIH (to Dr. Rawlings); and by funds from the Medical Research Council of Canada (to Dr. Afar).
We are indebted to Shirley Quan and Paulina Loo for expert technical assistance and to Julia Shimaoka and Kris Vensel for assistance in the preparation of the manuscript.
Source Information
From the Department of Microbiology and Molecular Genetics (D.C.S., D.J.R., D.E.H.A, O.N.W.) and the Howard Hughes Medical Institute (O.N.W.), University of California, Los Angeles; and the Department of Pediatrics, University of Tennessee College of Medicine, and the Department of Immunology, St. Jude Children's Research Hospital -- both in Memphis (O.P., M.E.F.-H., M.E.C.).
Address reprint requests to Dr. Conley at St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38101.
References
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Related Letters:
Atypical X-Linked Agammaglobulinemia
Kornfeld S. J., Good R. A., Litman G. W., Parolini O., Rohrer J., Conley M. E., Buckley R. H.
Extract |
Full Text
N Engl J Med 1994;
331:949-951, Oct 6, 1994.
Correspondence
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