Calcium regulates tertiary structure and enzymatic activity of human endometase/matrilysin-2 and its role in promoting human breast cancer cell invasion.
Human MMP-26 (matrix metalloproteinase-26) (also known as endometase or matrilysin-2) is a putative biomarker for human carcinomas of breast, prostate and other cancers of epithelial origin. Calcium modulates protein structure and function and may act as a molecular signal or switch in cells. The relationship between MMPs and calcium has barely been studied and is absent for MMP-26. We have investigated the calcium-binding sites and the role of calcium in MMP-26. MMP-26 has one high-affinity and one low-affinity calcium binding site. High-affinity calcium binding was restored at physiologically low calcium conditions with a calcium-dissociation constant of 63 nM without inducing secondary and tertiary structural changes. High-affinity calcium binding protects MMP-26 against thermal denaturation. Mutants of this site (D165A or E191A) lose enzymatic activity. Low-affinity calcium binding was restored at relatively high calcium concentrations and showed a K(d2) (low-affinity calcium-dissociation constant) value of 120 microM, which was accompanied with the recovery of enzymatic activity reversibly and tertiary structural changes, but without secondary structural rearrangements. Mutations at the low-affinity calcium-binding site (C3 site), K189E or D114A, induced enhanced affinity for the Ca2+ ion or an irreversible loss of enzymatic activity triggered by low-affinity calcium binding respectively. Mutation at non-calcium-binding site (V184D at C2 site) showed that C2 is not a true calcium-binding site. Observations from homology-modelled mutant structures correlated with these experimental results. A human breast cancer cell line, MDA-MB-231, transfected with wild-type MMP-26 cDNA showed a calcium-dependent invasive potential when compared with controls that were transfected with an inactive form of MMP-26 (E209A). Calcium-independent high invasiveness was observed in the K189E mutant MDA-MB-231 cell line.
Lee
Seakwoo
S
Department of Chemistry and Biochemistry and Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4390, USA.
Park
Hyun I
HI
Sang
Qing-Xiang Amy
QX
D000595:Amino Acid Sequence; D001483:Base Sequence; D001943:Breast Neoplasms / Q000201:enzymology* / Q000473:pathology*; D002118:Calcium / Q000502:physiology*; D017931:DNA Primers; D005260:Female; D006801:Humans; D053505:Matrix Metalloproteinases, Secreted / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009361:Neoplasm Invasiveness; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D011994:Recombinant Proteins; C410599:MMP26 protein, human; D053505:Matrix Metalloproteinases, Secreted; D002118:Calcium