Altered native stability is the dominant basis for susceptibility of α1-antitrypsin mutants to polymerization

Abstract

Serpins are protease inhibitors whose most stable state is achieved upon transition of a central 5-stranded β-sheet to a 6-stranded form. Mutations, low pH, denaturants and elevated temperatures promote this transition, which can result in a growing polymer chain of inactive molecules. Different types of polymer are possible, but, experimentally only heat has been shown to generate polymers in vitro consistent with ex vivo pathological specimens. Many mutations that alter the rate of heat-induced polymerization have been described, but interpretation is problematic because discrimination is lacking between the effect of global changes in native stability and specific effects on structural mechanism. We show that the temperature midpoint (Tm) of thermal denaturation reflects the transition of α1-antitrypsin to the polymerization intermediate, and determine the relationship with fixed-temperature polymerization half-times (t0.5) in the presence of stabilizing additives [TMAO (trimethylamine N-oxide), sucrose and sodium sulfate], point mutations and disulfide bonds. Combined with a retrospective analysis of 31 mutants characterized in the literature, the results of the present study show that global changes to native state stability are the predominant basis for the effects of mutations and osmolytes on heat-induced polymerization, summarized by the equation: ln(t0.5,mutant/t0.5,wild-type)=0.34×ΔTm. It is deviations from this relationship that hold key information about the polymerization process.

Update on alpha-1 antitrypsin deficiency: New therapies

“…Polymerisation also underlies the deficiency of the mild S (Glu264Val), I (Arg39Cys), Queen's (Lys154Asn) and Baghdad (Ala336Pro) alleles of α 1 -antitrypsin [12,14,19,20] but the rate of polymer formation is much slower in keeping with the absence of liver disease and only mild plasma deficiency. In many cases the reduction in the thermal stability of the native fold of α 1 -antitrypsin caused by mutations directly correlates with the polymerogenic tendency [11]. This implies that the mutants' conformational behaviour is qualitatively similar to that of wild-type α 1 -antitrypsin, but that destabilised states become accessible at lower temperatures.…”

“…We showed that the severe Z deficiency mutant of α 1 -antitrypsin is retained within the endoplasmic reticulum (ER) of hepatocytes as ordered polymers that become sequestered in Periodic Acid Schiff-positive, diastase-resistant inclusions [2,7]. We have used biophysical and crystallographic techniques to dissect the pathway of α 1 -antitrypsin polymerisation [8][9][10][11][12]. Our work suggests that the Z mutation perturbs its local environment (breach region, Fig.…”

“…Importantly one such exception is the Z variant, that appears to cause a relatively mild thermal destabilisation but is highly polymerogenic [11,12,16].…”

Deficiency Mutations of Alpha-1 Antitrypsin. Effects on Folding, Function, and Polymerization

“…Thermal denaturation experiments using SYPRO Orange involve an incremental increase in temperature (typically 1 8 C/min) and provide a measure of the stability of the native state of a protein, reported as the temperature at which a 50% "unfolding" is observed (T M ). For alpha-1 antitrypsin, this reports the unimolecular conversion from the native state to the polymerization intermediate (21).…”

“…Polymerization was monitored as the increase in acceptor (AF594) fluorescence at 615 nm with excitation of donor (AF488) at 470 nm using a Mastercycler realplex4 instrument (Eppendorf, Stevenage, UK) as described (17,21,22).…”