Homologous plasminogen N-terminal and plasminogen-related gene A and B peptides. Characterization of cDNAs and recombinant fusion proteins.
The cDNA corresponding to exons 2-4 of the processed human plasminogen (Pgn) gene, encoding the N-terminal peptide domain (NTP), has been cloned, expressed in Escherichia coli as a recombinant protein (r-NTP) containing a hexahistidine tag, and refolded to the native structure that contains two internal cystine bridges. RNA expression of the two Pgn-related genes, PRG A and PRG B, that potentially encode 9-kDa polypeptides having extensive similarity to the NTP has been investigated. Using RNA-based PCR with liver RNA as template, we demonstrate that PRG A encodes a detectable mRNA species. PRG A and PRG B have been found to be transcribed in the liver and yield virtually identical mRNAs. Neither of the PRGs are expressed in a variety of other normal tissues, as determined by Northern blot analysis. Factor-Xa digestion of the tagged r-NTP yields cleavage products which indicates that the expressed r-NTP domain of Pgn is endowed with a flexible conformation. Recombinant PRG B protein (r-PRG B) fused to a hexahistidine tag was purified and analyzed for structural integrity. Preliminary 1H-NMR spectroscopic data for r-NTP and r-PRG B indicate relatively fast amide 1H-2H exchange in 2H2O and close conformational characteristics for the two homologous polypeptides. Far ultraviolet-CD spectra for r-NTP and r-PRG B at pH 7.0 indicate similar defined secondary structure content for both domains, with 13-17% alpha-helix and 24-27% antiparallel beta-sheet. The fact that two transcriptionally active genes encode almost identical polypeptides supports the hypothesis that the Pgn NTP, together with the putative polypeptides encoded by the PRGs, may serve an important function, such as controlling the conformation of Pgn and thus its susceptibility to tissue activators.
Lewis
V O
VO
Orthopaedic Research Laboratories, Massachusetts General Hospital, Boston, USA.
Gehrmann
M
M
Weissbach
L
L
Hyman
J E
JE
Rielly
A
A
Jones
D G
DG
Llinás
M
M
Schaller
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010958:Plasminogen / Q000737:chemistry / Q000235:genetics*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic / Q000235:genetics
C118420:PLGLB1 protein, human; D010446:Peptide Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins; C118419:plasminogen-related protein A, human; D010958:Plasminogen; D015951:Factor Xa