Linked Life Data

18067325

Source:http://linkedlifedata.com/resource/pubmed/id/18067325

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2008-1-8
pubmed:abstractText
Human calcium-activated nucleotidase (CAN) exists as both a membrane-bound form in the endoplasmic reticulum and pre-Golgi intermediate membranes and as a secreted, soluble form. Although the wild-type human enzyme hydrolyzes ADP poorly, engineered soluble human proteins (SCANs) hydrolyze ADP much more efficiently, making them potentially useful therapeutic proteins for treatment of human clotting pathologies. According to the crystal structure and the recently identified dimeric nature of the soluble nucleotidase, the dimer interface contains a central core of hydrophobic residues. Previously, we demonstrated that the mutation of glutamic acid 130 (located in the dimer interface) to tyrosine increased both the tendency to form dimers and the ADPase activity. In the present study, we investigated the importance of the dimeric state for enzymatic activity and biological function in this nucleotidase by mutating isoleucine 170, which is located in the center of the hydrophobic core of the dimer interface. The results of analytical ultracentrifugation, chemical cross-linking, and tryptophan fluorescence analyses demonstrated that mutation of isoleucine 170 to either positively or negatively charged amino acids (lys or glu) disrupted the calcium-dependent dimerization in soluble CAN. Furthermore, these mutations decreased maximal ADPase activity for both the soluble and membrane-bound enzymes. Although not as critical as the hydrophobic interactions centered at isoleucine 170, the role of hydrophilic interactions in dimer formation was also demonstrated. Thus, mutation of aspartic acid 228 to threonine (D228T) decreased both the tendency to form dimers and ADPase activity, while double mutation of D228T/K224N largely restored the ability to form dimers and the ADPase activity, further indicating that the nucleotidase activity of CAN is linked to its quaternary structure. Since ADPase activity of the soluble form is crucial for its potential development as a therapeutic protein, these findings have implications for engineering the soluble human calcium-activated nucleotidase for clinical applications. In addition, future comparison of monomeric (I170K and I170E mutants) and dimeric (wild-type) crystal structures of SCAN will advance our understanding of its enzymatic mechanism and aid in engineering efforts.
pubmed:grant
pubmed:commentsCorrections
http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-10692345, http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-10694408, http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-12167635, http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-12234496, http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-12600208, http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-15006348, http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-15248776, http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-15608193, http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-16835225, http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-18404472, http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-3044186, http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-9675246, http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-9804829, http://linkedlifedata.com/resource/pubmed/commentcorrection/18067325-9890913
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
47
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
771-8
pubmed:dateRevised
2010-12-3
pubmed:meshHeading
pubmed:year
2008
pubmed:articleTitle
Characterization and importance of the dimer interface of human calcium-activated nucleotidase.
pubmed:affiliation
Department of Pharmacology and Cell Biophysics, University of Cincinnati, P.O. Box 670575, Cincinnati, Ohio 45267-0575, USA.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural