Characterisation of the complex of plasminogen activator inhibitor type 1 with tissue-type plasminogen activator by mass spectrometry and size-exclusion chromatography.
Glycosylated human plasminogen activator inhibitor type 1 (PAI-1), produced in Chinese hamster ovary (CHO) cells, showed a variety of compounds with different molecular weights when subjected to electrospray mass spectrometry (ES-MS), owing to the heterogeneity of the carbohydrate chains. However, non-glycosylated human PAI-1, produced in E. coli, gave rise to a prominent species with a molecular weight of 42,774, consistent with the amino-acid sequence. A non-glycosylated mutant of the proteinase domain (B-chain) of tissue-type plasminogen activator (tPA) produced in C 127 cells, had a molecular weight of 28,168. Full-length, glycosylated, tPA showed a large heterogeneity in molecular mass. For a mass study, a tPA-PAI-1 complex was formed, composed of non-glycosylated PAI-1 and non-glycosylated B-chain. This complex was remarkably stable at room temperature in buffer with a neutral pH. The mass spectrum of the complex provided two main species, a peptide with a mass of 3803 and a dominating species of 67,133. These masses are consistent with a complex where PAI-1 is cleaved at the P1-P1' position. A trace of a species with a molecular mass of 70,942 was also found, corresponding to the complete, non-dissociated complex with PAI-1. Separation of the cleaved peptide, corresponding to the hydrophobic C-terminal 33 amino-acid residues of PAI-1, from the complex, was achieved by size-exclusion chromatography in the presence of 30% acetonitrile. Thus, in the complex between tPA and PAI-1, the proteins are held together by a tight covalent bond, but the C-terminal cleaved peptide of PAI-1 is only bound to the complex by hydrophobic forces. To assess whether this is specific to the tPA B-chain alone, experiments with the complex of full-length, glycosylated tPA and glycosylated PAI-1 were also performed, and it was possible to demonstrate the release of the C-terminal PAI-1 peptide by chromatography, mass spectrometry, as well as by SDS-PAGE.
Strömqvist
M
M
Astra Hässle AB, Umeå, Sweden.
Karlsson
K E
KE
Björquist
P
P
Andersson
J O
JO
Byström
M
M
Hansson
L
L
Johansson
T
T
Deinum
J
J
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002850:Chromatography, Gel; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism
D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D010959:Tissue Plasminogen Activator