Unfolding of the immunoglobulin light and heavy chains is required for the enzymatic removal of N-terminal pyroglutamyl residues.
To enable Edman sequencing of pyroglutamylated immunoglobulins, enzymatic deblocking by pyroglutamate aminopeptidase is performed, often with variable yield and compromised solubility. Recently, enzymatic deblocking of immunoglobulins without denaturation was described. Although the conditions ensured efficient removal of pyroglutamyl residues, we conclude that deblocking is preceded by denaturation, which results in aggregation of the immunoglobulins. To study the effect of folding status on deblocking we developed a methanol based deblocking solution, which preserved the enzymatic activity of pyroglutamate aminopeptidase, provided conditions compatible with sequencing and enhanced deblocking of electroblotted samples, as well. At 50 degrees C and 35% (v/v) methanol the immunoglobulin chains were completely aggregated, but the degree of deblocking was comparable to that obtained with the previously described method. At 37 degrees C, the immunoglobulins were partly aggregated, but the deblocked chains were completely in the insoluble fractions, whereas the soluble fractions had retained pyroglutamylation in both chains, suggesting that unfolding of the immunoglobulins is required for the excision of the pyroglutamates. Inspection of the structures of pyroglutamylated immunoglobulin and pyroglutamate aminopeptidase P. furiosus indicates that the enzyme requires the substrate in an extended conformation, a criterium, which we conclude not to be fulfilled in the native form of immunoglobulins. Unfolding of the N-terminus would disrupt the immunoglobulin fold by breaking interactions between secondary structure elements and expose surfaces prone to aggregation.
Hellström
Johanna L
JL
University of Turku, Department of Biochemistry and Food Chemistry / Biotechnology, Tykistökatu 6 A, FIN-20520 Turku, Finland.
Vehniäinen
Markus
M
Mustonen
Merja
M
Lövgren
Timo
T
Lamminmäki
Urpo
U
Hellman
Jukka
J
D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism*; D007147:Immunoglobulin Light Chains / Q000378:metabolism*; D008958:Models, Molecular; D011489:Protein Denaturation; D020027:Pyrococcus furiosus / Q000201:enzymology; D011751:Pyroglutamyl-Peptidase I / Q000378:metabolism; D011761:Pyrrolidonecarboxylic Acid / Q000378:metabolism*
D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011751:Pyroglutamyl-Peptidase I; D011761:Pyrrolidonecarboxylic Acid