Studies of the interaction between human protein S and human C4b-binding protein using deletion variants of recombinant human protein S.
Human protein S interacts noncovalently with human C4b-binding protein (C4BP). We have studied this interaction using deletion variants of recombinant human protein S. Two deletion variants were constructed by restriction enzyme digestion and in vitro site-specific mutagenesis of the human protein S cDNA. The variants were stably expressed in C127 cells. Recombinant proteins were purified using Fast Flow Q anion-exchange chromatography. The activated protein C (APC) cofactor activity, C4BP binding properties and reactivity to different monoclonal antibodies against human protein S were examined. The first variant (E variant), which has a deletion of the third epidermal growth factor (EGF)-like domain (deletion of exon VII, corresponding to amino acid residues ASP-160 to Asp-202) expresses normal APC cofactor activity in a plasma system. This activity was inhibited by the addition of purified C4BP. The second variant (L variant), which has a deletion of the C-terminal loop of the sex hormone binding globulin (SHBG)-like domain (deletion of exon XV, corresponding to amino acid residues Asp-583 to Ser-635) also expresses normal APC cofactor activity in plasma. This activity could only be partially inhibited by the addition of purified C4BP. Binding of the recombinant proteins to C4BP was studied in a system using purified proteins. The E variant binds to C4BP with the same affinity similar as recombinant wild type protein S (apparent Kd approximately 10(-10) M). The L variant, however, shows a markedly reduced affinity for binding to C4BP (apparent Kd approximately 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)
Chang
G T
GT
Department of Haematology, University Hospital Utrecht, The Netherlands.
Maas
B H
BH
Ploos van Amstel
H K
HK
Reitsma
P H
PH
Bertina
R M
RM
Bouma
B N
BN
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D006801:Humans; D015674:Mammary Neoplasms, Animal; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011486:Protein C / Q000378:metabolism; D017293:Protein S / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D011486:Protein C; D017293:Protein S; D011993:Recombinant Fusion Proteins