Characterisation of the fibrinogenolytic properties of the buccal gland secretion from Lampetra japonica.
Lampetra japonica is representative of the ancient cyclostomota class of animals, and its buccal gland secretion (called lamphredin) is known to act as an anticoagulant. In this study, it was observed by both native-PAGE and SDS-PAGE that the secretion mainly contained two protein bands, buccal gland secretion protein-1 (BGSP-1, 159,909 Da) and buccal gland secretion protein-2 (BGSP-2, 25,660 Da). The N-terminal amino acids of BGSP-1 (EAESF QNLKT RICGG LNGLG) and BGSP-2 (TSVND WKLLD TKLSA NRKVI) were sequenced. Using a Sephadex G-75 column, we isolated BGSP-1, BGSP-2 and small peptides from the buccal gland secretion, but found only BGSP-1 showed fibrinogenolytic activity. BGSP-1 and lamphredin were found to rapidly degrade the alpha chain of human fibrinogen, slowly degrade the beta chain and hardly degrade the gamma chain. BGSP-1 and lamphredin showed a similar map by SDS-PAGE for the degradation of fibrinogen by cleavage at Ala(10)-Glu(11) and His(368)-Ser(369). BGSP-1 was also found to hydrolyze neuronal protein tau at Glu(12)-Asp(13) and Gln(244)-Thr(245). Further study showed that lamphredin and BGSP-1 were inactivated in the presence of a metal chelating agent EDTA. However, addition of Ca(2+) or Mg(2+) but not Zn(2+) restored the fibrinogenolytic activity. This suggests that BGSP-1 acts in the buccal gland as a metalloproteinase with a broad substrate specificity. Furthermore, the secretion showed cytolytic properties towards human SH-SY5Y and HeLa cells in culture, and lamphredin at a 50-fold dilution induced cell death.
Xiao
Rong
R
School of Life Sciences, Liaoning Normal University, Dalian 116029, China.
Li
Qing-Wei
QW
Perrett
Sarah
S
He
Rong-Qiao
RQ
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism*; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007798:Lampreys / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D045726:Metalloproteases / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016875:tau Proteins / Q000378:metabolism
D000925:Anticoagulants; D029941:Fish Proteins; D016875:tau Proteins; D005340:Fibrinogen; D045726:Metalloproteases; D008274:Magnesium; D002118:Calcium