Identification of trypsin-inhibitory site and structure determination of human SPINK2 serine proteinase inhibitor.
Human serine proteinase inhibitor Kazal-type 2 (SPINK2) functions as a trypsin/acrosin inhibitor and is synthesized mainly in the testis and seminal vesicle where its activity is engaged in fertility. The SPINK2 protein contains a typical Kazal domain composed by six cysteine residues forming three disulfide bridges. The expression of SPINK2 is closely related to cancer such as lymphomas, in that a high transcript level of SPINK2 in patients with primary cutaneous follicle center cell lymphomas have better prognosis with lower mortality. To clarify the role of SPINK2 in cancer, we performed quantitative real-time PCR and showed that the expression level of SPINK2 is significantly elevated in most leukemia cell lines except B-lymphoblast TK-6 cells. The molecular function and structural features of SPINK2 were also investigated by employing the recombinant active and mutant inactive SPINK2 proteins to determine its key P2-P2' (Pro(23)-Arg(24)-His(25)-Phe(26)) active site. The inhibition assay results demonstrated that Arg(24) at the P1 site is crucial for the specificity of SPINK2 on target enzyme. Although His(25) at the P1' and Phe(26) at the P2' residues are also involved in trypsin-SPINK2 interaction, Pro(23) at the P2 site may not be directly participated in interacting with trypsin. In addition, we determined the 3D solution structure of SPINK2 and used this structure to predict the SPINK2-proteinase complex structure and binding properties. These studies not only provide critical information about the structural properties and biophysical features of the SPINK2 proteinase inhibitor, but also suggest its important role in tumor progression and response to treatment.
Chen
Ting
T
Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan.
Lee
Tian-Ren
TR
Liang
Wei-Guang
WG
Chang
Wun-Shaing Wayne
WS
Lyu
Ping-Chiang
PC
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000074162:Serine Peptidase Inhibitors, Kazal Type
D006023:Glycoproteins; D011994:Recombinant Proteins; C066238:SPINK2 protein, human; D000074162:Serine Peptidase Inhibitors, Kazal Type