title: The C-terminal domains of the gingipain K polyprotein are necessary for assembly of the active enzyme and expression of associated activities.
abstract: The Porphyromonas gingivalis lysine-specific cysteine protease (gingipain K, Kgp) is expressed as a large precursor protein consisting of a leader sequence, a pro-fragment, a catalytic domain with a C-terminal IgG-like subdomain (IgSF) and a large haemagglutinin/adhesion (HA) domain. In order to directly study the role of these non-catalytic domains in pro-Kgp processing and maturation in P. gingivalis, the wild-type form of the gene was replaced with deletion variants encoding C-terminally truncated proteins, including KgpDeltaHA3/4 (Delta1292-1732 aa), KgpDeltaHA2-4 (Delta1157-1732 aa), KgpDeltaHA1-4 (Delta738-1732 aa), KgpDeltaC-term/HA (Delta681-1732 aa) and KgpDeltaIg/C-term/HA (602-1732 aa). Northern blot and reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that all truncated variants of the kgp gene were transcribed in P. gingivalis. Despite high levels of kgpDeltaC-term/HA and kgpDeltaIg/C-term/HA transcripts, no Kgp-specific antigen was detected in cultures of these mutants as determined by Western blot analysis with monoclonal antibodies specific for the Kgp catalytic domain. Furthermore, only barely measurable amounts of Kgp-specific activity were detected in these two mutants. The remaining mutants expressed significant Kgp activity, however, at lower levels when compared with the parental strain. The decreased activity most probably resulted from altered folding and/or hindered secretion of the protein. The kgp gene truncation was also demonstrated to alter the distribution of the gingipain protein between membrane-associated and -secreted forms. While both gingipain K activity and the protein were cell membrane-associated in the parental strain, the mutants released significant amounts of both protein and activity into the media. Taken together, these results suggest that the C-terminal HA domains of Kgp are not only essential for full expression of gingipain activity, but also for proper processing of the multiprotein complex assembly on the P. gingivalis outer membrane. Moreover, our results indicate that the immunoglobulin-like subdomain is indispensable for proper folding and expression of the gingipains.
authors.affiliation: Department of Microbiology, Faculty of Biotechnology, Jagiellonian University, Kraków, Poland.
authors.forename: Maryta
authors.initials: M
authors.lastname: Sztukowska
authors.forename: Aneta
authors.initials: A
authors.lastname: Sroka
authors.forename: Marcin
authors.initials: M
authors.lastname: Bugno
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Banbula
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Robert N
authors.initials: RN
authors.lastname: Pike
authors.forename: Caroline A
authors.initials: CA
authors.lastname: Genco
authors.forename: James
authors.initials: J
authors.lastname: Travis
authors.forename: Jan
authors.initials: J
authors.lastname: Potempa
mesh_terms: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D015152:Blotting, Northern; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry; D003470:Culture Media / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D005798:Genes, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014158:Transcription, Genetic
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D003470:Culture Media; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D003546:Cysteine Endopeptidases