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A protease‐sensitive site in the proposed Ca 2+ ‐binding region of human serum amyloid P component and other pentraxins
Author(s) -
Kinoshita Carol M.,
Gewurz Anita T.,
Siegel Joan N.,
Ying ShanChing,
Gupta Rajesh K.,
Huckman Robert,
Gewurz Henry,
Hugli Tony E.,
Coe John E.
Publication year - 1992
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560010602
Subject(s) - chymotrypsin , serum amyloid p component , proteolysis , proteases , pronase , trypsin , biochemistry , chemistry , protease , binding site , proteinase k , gel electrophoresis , microbiology and biotechnology , biology , enzyme , c reactive protein , immunology , inflammation
Serum amyloid P component (SAP) is a decamer of 10 identical 25.5‐kDa subunits. Limited proteolysis of SAP with α‐chymotrypsin cleaves the subunit into two fragments of 18 and 7.5 kDa, although the fragments stay together in the decamer under nondenaturing conditions. Proteolysis does not occur in the presence of Ca 2+ (10 mM). Cleavage with α‐chymotrypsin prevents the Ca 2+ ‐dependent binding of SAP to zymosan extract, nucleosomes, and DNA. The α‐chymotrypsin cleavage site identified is in a region of SAP that is highly conserved in members of the human C‐reactive protein (CRP) family of proteins (pentraxins) to which SAP belongs and is similar to the Ca 2+ ‐binding site in calmodulin and related Ca 2+ ‐binding proteins (Nguyen, N.Y., Suzuki, A., Boykins, R.A., & Liu, T.‐Y., 1986, J. Biol. Chem. 261 , 10456–10465). Treatment of SAP with other proteases (trypsin, Pronase, and Nagarse protease) yields fragmentation patterns upon sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) that are similar to those obtained with α‐chymotrypsin. Two other members of the pentraxin family of proteins, hamster female protein and rabbit CRP, also exhibit similar fragmentation patterns on SDS‐PAGE when treated with the various proteases. Recently, it has been shown that the homologous protein, human CRP, is cleaved in the same homologous position as cleavage of SAP by α‐chymotrypsin, resulting in the loss of Ca 2+ binding (as shown by equilibrium dialysis) and Ca 2+ ‐dependent binding reactivities (Kinoshita, C.M., Ying, S.‐C., Hugli, T.E., Siegel, J.N., Potempa, L.A., Jiang, H.J., Houghten, R.A., & Gewurz, H., 1989, Biochemistry 28 , 9840–9848). These results indicate that the protease sensitivity of this proposed Ca 2+ ‐binding region has been conserved and may play an important regulatory role, perhaps via the control of Ca 2+ ‐dependent properties of these proteins.