Characterization of monoclonal antibodies to calpain 3 and protein expression in muscle from patients with limb-girdle muscular dystrophy type 2A

Abstract

Monoclonal antibodies were raised to two regions of calpain 3 (muscle-specific calcium-activated neutral protease), which is the product of the gene that is defective in limb-girdle muscular dystrophy type 2A. The antibodies produced characteristic patterns of bands on Western blots: normal calpain 3 protein was represented by bands at 94 kd, plus additional fragments at approximately 60 or 30 kd, according to the antibody used. Specificity was confirmed by the loss of all bands in patients with null gene mutations. The "normal" profile of bands was observed in muscle from 33 control subjects and 70 disease-control patients. Calpain 3 protein was found to be extremely stable in fresh human muscle, with full-size protein being detected 8 hours after the muscle had been removed. Blots of muscle from nine limb-girdle muscular dystrophy type 2A patients with defined mutations showed variation in protein expression, with seven showing a clear reduction in the abundance of protein detected. No simple relationship was found between the abundance and clinical severity. Two patients showed normal expression of the full-size 94 kd band accompanied by a clear reduction in the smaller fragments. This pattern was also observed in one patient with an undefined form of limb-girdle dystrophy. These results indicate that immunodiagnosis is feasible, but caution will need to be exercised with the interpretation of near-normal protein profiles.

Figure 1.

Three strips from a Western blot of human skeletal muscle labeled with different monoclonal antibodies (indirect peroxidase). On the right is an expanded view of the bands in the region of 78 to 94 kd. Approximate molecular masses for the principal bands of interest are indicated as a guide on the left.

Figure 2.

Samples of skeletal muscle from different animals analyzed on Western blots labeled with exon 1 antibody Calp3d/2C4 (top), exon 8 antibody Calp3c/11B3 (middle), or exon 8 antibody Calp3d/12A2 (bottom). The samples are as follows: Ms, mouse; Rt, rat; Rb, rabbit; Dg, dog; Ch, chicken; Ha, hamster; Pg, pig; and Hu, human. Approximate molecular masses are shown as a guide on the right.

Figure 3.

Western blots of samples from selected time points in three muscle breakdown experiments, labeled with exon 1 antibody Calp3d/2C4 (top) or exon 8 antibody (bottom). A, time before freezing fresh muscle; B, time frozen muscle was left to thaw before homogenization; C, time as homogenate in saline before the addition of double-strength electrophoresis treatment buffer. In C, only half the amount of muscle protein was loaded compared with A and B (see Materials and Methods). For each experiment: T₀ = immediately or at the earliest time point possible; h, hours; m, minutes. Approximate molecular masses are shown as a guide on the right.

Figure 4.

Twenty lanes of control and patient biopsies analyzed on Western blots labeled with exon 1 antibody Calp3d/2C4 (top) or exon 8 antibody Calp3c/12A2 (bottom). Each panel contains lanes from three different blots, as indicated by the vertical lines. Lanes 1, 3, 5, 17, and 20: Different normal control samples. Lanes 2 , 4, 6, 8, 10, 11, 13, 18, and 19: patients with LGMD2A (see Table 2 ▶ ). The other lanes are from patients with different types of MD: lane 7, merosin-deficient congenital MD; lane 9, facioscapulohumeral MD; lane 12, γ-sarcoglycanopathy (LGMD2C); lane 14, Becker MD; lane 15, undefined limb-girdle MD (?LGMD); and lane 16, Duchenne MD. Approximate molecular masses are shown as a guide on the right.

Figure 5.

Comparison of the amino acids in the two human calpain 3 peptide sequences used to raise monoclonal antibodies. Amino acids 1 to 19 only occur in calpain 3, but amino acids 355 to 370 have corresponding sequences in calpains 2 and 1, as indicated in the (top). Only the amino acids that differ are indicated; a dash means that the amino acids are the same. Bottom: The human sequences in calpains 3, 2, and 1 are compared with the corresponding sequences in different animals. The first row depicts the human sequences, and subsequent rows show any differences in the corresponding amino acids in mouse, rat, pig, and chicken. Sequences are not currently available for pig calpains 2 and 1 or for calpains 3, 2, and 1 in the other species examined on blots.