Abstract:
:To identify functionally essential sequences and residues of CTLA-2alpha, in vitro mutagenesis was carried out. The coefficient of inhibition (K(i)) was determined towards rabbit cathepsin L using Z-Phe-Arg-MCA as the substrate. Recombinant CTLA-2alpha inhibited the enzyme potently (K(i) = 15 nM). A truncated mutant, lacking the N- and C-terminal Ala1-Asp9 and Leu80-Glu109 regions, was also a potent inhibitor (K(i) = 10 nM). Subsequent short deletions in the central region (Asn10-Ser79) showed three functionally essential distinct regions: Asn10-Phe19, His30-Ala44 and Ser55-Ser79. These regions cover sequences corresponding to three helices (alpha1, alpha2 and alpha3) and sequences that interact with the cognate enzyme. Alanine scanning showed that replacement of one of three conserved Trp residues increased the K(i) by 15-20-fold; whereas, replacement of two/three Trp residues at once caused complete loss of potency, as did replacing Cys75 with Ala or Ser. The proteins from wild-type (WT) CTLA-2alpha and mutant C75A were stable overnight when incubated with cathepsin L; whereas, proteins from mutants W12A, W15A and W35A were quickly digested. Incubation of cathepsin L/WT CTLA-2alpha formed a complex; whereas, C75S did not form a complex. Our overall results point to a critical role of W12, W15, W35 and Cys75 residues in CTLA-2alpha.
journal_name
J Biochemjournal_title
Journal of biochemistryauthors
Deshapriya RM,Yuhashi S,Usui M,Kageyama T,Yamamoto Ydoi
10.1093/jb/mvp188subject
Has Abstractpub_date
2010-03-01 00:00:00pages
393-404issue
3eissn
0021-924Xissn
1756-2651pii
mvp188journal_volume
147pub_type
杂志文章abstract::Phospholipase A2 inhibitor (PLI) was purified from the blood plasma of Chinese Mamushi, Agkistrodon blomhoffii siniticus, by sequential chromatography on Sephadex G-200, Mono Q, and Blue-Sepharose CL-6B columns. The purified PLI was a glycoprotein with an apparent molecular mass of 75 kDa and was composed of a single ...
journal_title:Journal of biochemistry
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a124767
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journal_title:Journal of biochemistry
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doi:10.1093/oxfordjournals.jbchem.a123982
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a133874
更新日期:1982-05-01 00:00:00
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pub_type: 杂志文章
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a131394
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a134061
更新日期:1982-11-01 00:00:00
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pub_type: 杂志文章
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更新日期:2005-03-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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更新日期:2004-02-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:
更新日期:1981-04-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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更新日期:2009-07-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a021500
更新日期:1996-11-01 00:00:00
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pub_type: 杂志文章
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更新日期:2006-06-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a134719
更新日期:1984-04-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a022732
更新日期:2000-07-01 00:00:00
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pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a135367
更新日期:1985-10-01 00:00:00
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pub_type: 杂志文章
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更新日期:2020-10-01 00:00:00
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pub_type: 杂志文章
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pub_type: 杂志文章
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更新日期:1984-03-01 00:00:00
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pub_type: 杂志文章
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pub_type: 杂志文章
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更新日期:1987-01-01 00:00:00
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pub_type: 杂志文章
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更新日期:2000-12-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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更新日期:1994-05-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a130799
更新日期:1975-04-01 00:00:00
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更新日期:2009-01-01 00:00:00
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pub_type: 杂志文章
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更新日期:2003-08-01 00:00:00