Hereditary spastic paraplegia SPG13 is associated with a mutation in the gene encoding the mitochondrial chaperonin Hsp60

Abstract

SPG13, an autosomal dominant form of pure hereditary spastic paraplegia, was recently mapped to chromosome 2q24-34 in a French family. Here we present genetic data indicating that SPG13 is associated with a mutation, in the gene encoding the human mitochondrial chaperonin Hsp60, that results in the V72I substitution. A complementation assay showed that wild-type HSP60 (also known as "HSPD1"), but not HSP60 (V72I), together with the co-chaperonin HSP10 (also known as "HSPE1"), can support growth of Escherichia coli cells in which the homologous chromosomal groESgroEL chaperonin genes have been deleted. Taken together, our data strongly indicate that the V72I variation is the first disease-causing mutation that has been identified in HSP60.

Figure 1

Pedigree of family SAL-612. SPG13 is flanked by D2S2195 and D2S309. The haplotype segregating with HSP is indicated, and recombinants are noted by an arrow. The plus sign identifies the individuals in whom the HSP60 292 G→A variant was found. A perfect segregation is observed between the disease haplotype and the HSP60 292 G→A variant.

Figure 2

Schematic representation of the structure of the Hsp60 chaperonin homologue GroEL from E. coli (Protein Data Bank entry 1AON). The architecture of a subunit in the closed and open conformations and an overview of the GroEL/GroES chaperonin complex with one subunit in the closed (upper ring) and open (lower ring) conformations highlighted in black are shown. The side chains of valine-74 and lysine-160, which correspond to valine-72 and asparagine-158, respectively, in human Hsp60, are shown in space-filling representation. The figure was produced with WebLab ViewerLite software (Molecular Simulations).

Figure 3

Western blot analysis of Hsp60 and GroEL protein expression. E. coli B178 cells transformed with the plasmids carrying the cDNA for human HSP10 and wt or variant HSP60 (see table 1) or cells transformed with the vector plasmid were grown in dYT medium to midlog phase. Expression of plasmid-encoded chaperonins was induced for 3 h by addition of 1 mM IPTG. One of the groESgroEL deleted colonies that was maintained alive with plasmid pOFX HSP60 (wt)–HSP10 (wt) was grown overnight in dYT medium that was supplemented with 1 mM IPTG. Soluble extracts prepared as described elsewhere (Bross et al. 1995) were subjected to SDS-PAGE and western blot analysis through use of monoclonal antibodies that were directed against Hsp60 (H-3524; Sigma) and GroEL (SPA 870; StressGen), respectively. Blots were developed using ECL⁺ (Amersham Biosciences) and were scanned using a STORM molecular imager. The strain genotypes with respect to chromosomal groESgroEL and plasmid-expressed human Hsp60 variant and the amount of total protein loaded per lane are indicated at the top. The position and molecular mass (in kD) of co-electrophoresed marker proteins are indicated at the right margin.