Asparagine deamidation in recombinant human lymphotoxin: hindrance by three-dimensional structures.
The chemical stability of recombinant human lymphotoxin (rhLT) was evaluated at pH 7, 9, and 11 and 40 degrees C using quantitative tryptic map and urea-IEF methods. Degradation products were characterized by mass spectrometry. The stability of denatured rhLT protein was also evaluated to elucidate the effects of three-dimensional structures on Asn deamidation in rhLT. Two sites that underwent Asn deamidation were identified in rhLT, Asn(19) and Asn(40)-Asn(41). At pH 11 and 40 degrees C, deamidation at Asn(19) and Asn(40)-Asn(41) had half-lives of 14 +/- 4 and 80 +/- 24 days, respectively. Upon denaturation, 31- and ninefold acceleration in the degradation rates was observed at the Asn(19) and Asn(40)-Asn(41) sites, respectively. The rate of Asn(19) degradation in denatured rhLT was comparable to that of the model peptide possessing the same primary sequence as the Asn(19)-containing region in rhLT. Analysis of the rhLT crystal structure revealed that both Asn deamidation sites were located in beta-turn structures with extensive hydrogen-bonding networks created with nearby residues in the tertiary structures. The results suggested that these tertiary and secondary structures, if held true in solution, were probably responsible for the stabilization of Asn in the native rhLT protein by reducing flexibility, thus preventing adoption of the favorable conformation required for cyclic-imide formation.
Xie
Minli
M
Bristol-Myers Squibb Company, Pharmaceutical Research Institute, Rt. 141 and Henry Clay Road, Wilmington, Delaware 19880, USA. minli.xie@bms.com
Shahrokh
Zahra
Z
Kadkhodayan
Miryam
M
Henzel
William J
WJ
Powell
Michael F
MF
Borchardt
Ronald T
RT
Schowen
Richard L
RL
D001216:Asparagine / Q000737:chemistry*; D002942:Circular Dichroism; D003460:Crystallization; D004355:Drug Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008233:Lymphotoxin-alpha / Q000737:chemistry*; D013058:Mass Spectrometry; D008958:Models, Molecular; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D008233:Lymphotoxin-alpha; D001216:Asparagine