Purification and cDNA cloning of a novel protease inhibitor secreted into culture supernatant by MDCK cells.
The infectivity of influenza viruses to host cells depends on the activation of the viral glycoprotein hemagglutinin (HA) by proteases. Starting from the observation that influenza virus replication in MDCK (Madin Darby canine kidney) cells was impaired by inactivation of trypsin in the culture fluids, we demonstrated that the inhibitory activity was resolved into two Trypsin-inactivating factors (TF), TF A (15 kDa) and TF B (11 kDa). N-terminal protein sequences of the factors revealed that TF A was a known Submandibular Protease Inhibitor (SPI) secreted in dog saliva, while TF B was a novel protein (renamed CKPI; canine kidney protease inhibitor). Following peptide mapping and protein sequencing of CKPI we obtained a 390 bp cDNA encoding a 130-amino-acid protein from MDCK cell total RNA. Protein sequence comparison showed a 63.8% identity with human secretory leukocyte protease inhibitor (SLPI), the molecule containing two conserved whey acidic protein (WAP) motifs, and we suggest that CKPI is thought to be the canine analogue of human SLPI. These results suggest that the inhibitory factors are secreted from MDCK cells, which are involved in prevention of virus replication, and applicable to the protection of host cells from virus infection.
Nishiyama
Kiyoto
K
Applied Research Department, Kikuchi Research Center, The Chemo-Sero-Therapeutic Research Institute, Kyokushi Kawabe 1314-1, Kikuchi, Kumamoto 869-1298, Japan. nishiyama-ki@kaketsuken.or.jp
Sugawara
Keishin
K
Nouchi
Toshinobu
T
Kawano
Nami
N
Soejima
Kenji
K
Abe
Shin-Ichi
S
Mizokami
Hiroshi
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification*; D004285:Dogs; D006168:Guinea Pigs; D006801:Humans; D009980:Influenza A virus / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D014361:Trypsin Inhibitors / Q000302:isolation & purification / Q000378:metabolism; D014709:Vero Cells; D014775:Virus Activation / Q000502:physiology
D018076:DNA, Complementary; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors