Protein Stability, Folding/Unfolding, and Formation of D

Investigators

Abstract

Our primary research project involves A1-PI, which is a physiologically important serine protease inhibitor. The folding mechanisms of A1-PI are under investigation in order to elucidate the relationship between its structural stability and physiological function and, thus, its tendency and the substantially increased tendency of certain natural mutants to polymerize. Studies of guanidine- and thermally-induced unfolding and refolding of the normal form and the most common variant of a1-PI will enhance our understanding of this class of inhibitors and allow the development of strategies for stabilizing the physiologically active form of this molecule thereby minimizing polymerization. Potency of A1-PI is commonly determined by measuring the extent of inhibition of the steady-state activity of trypsin, a serine protease, the active concentration of which has been established. In order to minimize effects of trypsin autodigestion, we are investigating the appropriate steady-state substrate and trypsin concentrations to use for routine assays. Recently, we have cloned a normal variant of A1-PI into E. coli to assess the feasibility of doing the same for the most common mutant A1-PI, the Z mutant, associated with drastically reduced circulating levels due to polymerization within hepatocytes, the site of synthesis, presumably during prolonged folding of this mutant. The recombinant Z form will augment the limited amount of natural mutant available and allow an assessment of the role glycosylation plays in protein stabilization and tendency to polymerize. The other research project concerns the development of a facile, accurate SE-HPLC method for determining the weight average and number average molecular weights of Hetastarch, a highly polydisperse HES, which is used as a non-protein colloidal plasma volume expander akin to the method utilized in the USP monographs for Dextran 40 and Dextran 70. Such molecular weight data are used to access the oncotic potential. Protein Stability, Folding/Unfolding, and Formation of Deleterious Protein Species. Human alpha-1-proteinase inhibitor (A1-PI) is a naturally occurring serine protease inhibitor, the normal function of which is to inhibit neutrophil elastase primarily in lung tissue. Patients homozygous for the Z mutant form of A1-PI suffer from progressive emphysema and have an increased risk for the development of liver disease because of the accumulation in hepatocytes, the site of synthesis, of inclusion bodies containing linear polymers of A1-PI thereby resulting in substantially reduced circulating levels with particularly deleterious effects on lung tissue due to the action of uninhibited elastase. The tendency of the Z mutant to polymerize has been attributed to its slower folding during translation. The most widely accepted model of polymerization proposes that a linear, head-to-tail polymer forms by sequential insertion of the reactive center loop (RCL) of one A1-PI monomer between the central strands of the A beta-sheet of an adjacent monomer. This model derives primarily from two observations: peptides that are homologous with the RCL insert into the A beta-sheet of the A1-PI monomer and this insertion prevents A1-PI polymerization. Normal A1-PI monomer does not spontaneously polymerize; however, we have shown that the disulfide-linked dimer of normal A1-PI spontaneously forms linear polymers in buffer. The monomers within this dimer are joined head-to-head. Thus, the arrangement of monomers in these polymers must be different from that predicted by the loop-A sheet model. As a result, we have proposed a new model for A1-PI polymer. In addition, polymerization of disulfide-linked dimer is not inhibited by the presence of the peptide even though dimer appears to interact with the peptide. Thus, RCL insertion into A beta-sheets may not occur during polymerization of this dimer. The generation of an A1-PI reference standard, required for assaying potency, initially involves active site titration of a sample of trypsin to determine its active concentration. The potency of an A1-PI reference standard is determined by measuring the molar ratio of the concentration of A1-PI required to exactly inhibit the steady-state activity of a known concentration of active trypsin. The A1-PI reference standard is used to establish a standard curve for the inhibition of the steady state activity of trypsin or an elastase thereby allowing the determination of the inhibitory activity of a sample of A1-PI. Difficulties often observed with this approach involve the steady state activity of trypsin becoming non-linear at higher trypsin concentrations and measured molar ratios of active A1-PI to active trypsin greater than unity for exact inhibition of trypsin, even though the stoichiometry of inhibition should be unity. These effects relate to the competitive inhibition of trypsin itself with the steady state substrate and with A1-PI. We are currently involved in a systematic approach by investigating these effects with different lots of trypsin from different sources and with steady state substrates for which trypsin has substantially different affinities (i.e., different Km values). A long-range goal is to participate in the establishment of a WHO A1-PI standard. The gene for a normal A1-PI variant that had been incorporated into a vector designed for use in a clinical gene therapy study was obtained from a clinical research laboratory. The gene was excised from this vector and inserted into a commercial vector in which it was attached to a histidine tag. The E. coli Rosetta strain was transformed with this plasmid then grown and harvested. In a preliminary experiment, A1-PI was detected antigenically in cell lysate and in a newly, partially purified preparation (purified with a nickel, histidine binding column). Data suggest that protease activity must be inhibited in the cell lysate. Development of the purification process continues. Characterization of Non-Protein Colloidal Plasma Volume Expanders. Several years ago, monographs for Dextran 40 and Dextran 70 drug substances and products were adopted by the USP. A SE-HPLC methodology is used to determine weight average and number average molecular weights (Mw and Mn, respectively) of these polydisperse materials in conjunction with Dextran molecular weight standards for which accurate Mw values are known. Mw and Mn values of a lot of such material provide a measure of oncotic potential (or potency). Calibration of the HPLC set-up is performed by the assignment of monodisperse molecular weights to each of the retention times recorded by performing a simultaneous non-linear least squares fit of the elution profiles of the Dextran molecular weight standards with a compiled program provided by a Dextran manufacturer thereby permitting the compuation of Mw and Mn for a Dextran sample from its elution profile. The behavior of this program is sometimes problematic. In an effort to ameliorate this situation with regard to the Dextrans, but also with respect to a similar approach for determining Mw and Mn for Hetastarch, we have just started to investigate whether a listing of a Fortran 70 version of this program can be compiled and used. If successful, an attempt to write a Basic version of this program will be made in order to have both interpretive and compiled versions available for use (and modification to facilitate its use, if necessary). This is the first step in attempting to develop a USP monograph for Hetastarch. This project incorporates FY2002 projects 1Z01BQ004004-10 and 1Z01BQ004005-10.