Polyamine-promoted autoactivation of plasma hyaluronan-binding protein.
BACKGROUND
Plasma hyaluronan-binding protein (PHBP), a protease implicated in extracellular proteolysis, consists of multiple domains: an N-terminal region (NTR), three epidermal growth factor (EGF)-like domains, a kringle domain, and a protease domain. PHBP circulates as a single-chain proenzyme (pro-PHBP), which is converted to an active, two-chain form through autoproteolysis.


OBJECTIVE
To understand the mechanism of autoactivation. Here, we report that polyamine induces the formation of pro-PHBP autoactivation complex, in which an intermolecular interaction between NTR and the third EGF-like domain (E3) plays a role.


METHODS
Using a series of pro-PHBP mutants that partially lack functional domains, polyamine-induced pro-PHBP autoactivation was investigated in terms of enzyme activity, protein interaction, and inhibition by carminic acid, an anthraquinone compound identified in this study.


RESULTS
Polyamine enhanced intermolecular binding of pro-PHBP, but not of mutant pro-PHBP that partially lacked NTR (DeltaN). Carminic acid inhibited intermolecular pro-PHBP binding and specifically abolished polyamine-induced autoactivation. NTR bound to pro-PHBP and DeltaN, but its binding was minimal to a mutant that lacked E3. The NTR-DeltaN binding was inhibited by a combination of polyamine and carminic acid, but each compound alone was ineffective.


CONCLUSIONS
We infer from the data that (i) polyamine modulates intramolecular NTR-E3 interaction to allow intermolecular binding between NTR and E3 in another pro-PHBP molecule to form an autoactivation complex, and (ii) carminic acid inhibits polyamine-modulated intermolecular NTR-E3 binding. Polyamine concentrations are higher in cells and tissues with inflammation and malignancy. Polyamine leakage from legions through cell death or tissue injury may account for physiologically relevant pro-PHBP activation.
Yamamichi
S
S
Department of Applied Biological Science, Tokyo Noko University, 3-5-8 Saiwaicho, Fuchu-shi, Tokyo 183-8509, Japan.
Nishitani
M
M
Nishimura
N
N
Matsushita
Y
Y
Hasumi
K
K
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001777:Blood Coagulation; D002329:Carmine / Q000031:analogs & derivatives / Q000737:chemistry; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D006801:Humans; D008546:Melanoma, Experimental; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011073:Polyamines / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011700:Putrescine / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013095:Spermidine / Q000378:metabolism; D013096:Spermine / Q000378:metabolism; D014162:Transfection
D011073:Polyamines; D013096:Spermine; D002329:Carmine; C433550:HABP2 protein, human; D012697:Serine Endopeptidases; D013095:Spermidine; D011700:Putrescine