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Case Reports
. 1999 Jul 20;96(15):8693-8.
doi: 10.1073/pnas.96.15.8693.

A heterozygous mutation of beta-actin associated with neutrophil dysfunction and recurrent infection

H Nunoi  1 , T Yamazaki, H Tsuchiya, S Kato, H L Malech, I Matsuda, S Kanegasaki
Affiliations
Case Reports

A heterozygous mutation of beta-actin associated with neutrophil dysfunction and recurrent infection

H Nunoi et al. Proc Natl Acad Sci U S A. .

Abstract

A human disorder caused by mutation in nonmuscle actin has not been reported. We report here a variant of nonmuscle actin in a female patient with recurrent infections, photosensitivity, and mental retardation. She also had abnormalities in neutrophil chemotaxis, superoxide production, and membrane potential response. Two-dimensional PAGE analysis of proteins from neutrophils and other cell types from this patient demonstrated a unique protein spot migrating at 42 kDa with pI shifted slightly to neutral relative to normal beta- and gamma-actin. Digestion peptide mapping and Western blotting showed this spot to be an abnormal actin. A full-length cDNA library was constructed by using mRNA from patient's cells and cDNA encoding the mutant beta-actin molecule was identified by an in vitro translation method. Sequencing of the clones demonstrated a G-1174 to A substitution, predicting a glutamic acid-364 to lysine substitution in beta-actin and eliminating a HinfI DNase restriction site found in normal beta-actin sequence. By HinfI digestion and by sequencing, the mutation in one allele of patient's genomic DNA was confirmed. Though no defect in cell-free polymerization of actin was detected, this defect lies in a domain important for binding to profilin and other actin-regulatory molecules. In fact, the mutant actin bound to profilin less efficiently than normal actin did. Heterozygous expression of mutant beta-actin in neutrophils and other cells of this patient may act in a dominant-negative fashion to adversely affect cellular activities dependent on the function of nonmuscle actin.

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Figures

Figure 1
Figure 1
Functional defects in neutrophils from the patient. (A) Chemotactic ability assessed by Boyden chamber method toward 10−8 (1) or 10−9 M fMLP (2) or 10% zymosan-activated serum (3). Vertical numbers indicate numbers of cells in a field. We used different concentrations of fMLP because we found previously that concentrations of fMLP necessary for maximum chemotactic ability were different between the cells from cord and adult blood. Chemotactic ability of cord blood neutrophils was 70–80% of normal adults at an optimum concentration of fMLP for adult cells. In each concentration, the cells from the patient exhibited significant chemotaxis deficiency even compared with the ability of the cells from cord blood. (B) Maximum rate of superoxide generation assayed on exposure to 2 × 10−7 M fMLP (1), 2 × 10−7 M fMLP plus 2.5 μg/ml cytochalasin D (2), or 2.5 μg/ml cytochalasin D plus 50 μg/ml Con A (3). Vertical numbers indicate nanomoles of superoxide/minute/106 neutrophils. Open column, patient; closed column, control. (C) Relative change of fluorescence as an indication of depolarization and repolarization of patient (Left) and control neutrophils (Right). Di-O-C5 [3] was used as the lipophilic probe, and 2 × 10−7 M fMLP was used as a stimulus (arrows). Neutrophils immediately after isolation (a) and those stored on ice for 1 hour (b) were used.
Figure 2
Figure 2
Analysis of proteins from the patient’s (A) and normal control neutrophils (B) with 2D PAGE. Closed arrows indicate β- (left) and γ-actin (right), and an open arrow indicates a unique spot. Molecular weight markers are indicated on the left.
Figure 3
Figure 3
In vitro translation products of actin cDNAs cloned from patient’s cells and normal control cells. Products of control β-actin (A), γ-actin (B), and the mutant β-actin cDNAs together with control β-actin molecules (C) were subjected to 2D PAGE. Closed and open arrows indicate products of normal and mutant actin cDNA, respectively. Each set of spots includes both nonacetylated (right) and acetylated actin (left). Molecular weight markers are indicated on the left.
Figure 4
Figure 4
Sequences around 1,174 of the actin cDNAs. The sense sequences and predicted amino acid sequences of control (Upper) and patient’s specimen (Lower) are shown.
Figure 5
Figure 5
HinfI digestion profile of PCR-generated, 405-bp fragment of β-actin gene from the patient (Pt) and Control (C1–3). Arrows from top to bottom indicate the position of 405-, 340-, and 65-bp fragments in this order. Mobility of each marker is indicated on the right.
Figure 6
Figure 6
Copolymerization of mutant and normal actin molecules from the patient’ B cell line. The cytosolic fraction from [35S]methionine-labeled patient’s cell (A) were polymerized and depolymerized as described in the text. The procedures were repeated three times, and, each time after centrifugation, the precipitate was analyzed by 2D PAGE (B–D). Open arrows indicate the mutant actin.
Figure 7
Figure 7
Binding of actin to profilin. The 35S-labeled translation products of normal and mutant actin cDNAs were incubated with the GST-profilin-beads or GST-beads at 4°C for 2 hours. Recovered labeled actin from the beads was subjected to SDS/PAGE. The gel was dried and exposed to Fuji imaging plate, and radioactivity of the actin band was determined by using Bas 2000 (Fuji). The values after subtraction of those obtained by using GST-beads (<10%) are plotted. Open circle, control β-actin; closed circle, mutant β-actin.
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