Identification of Functionally Important Residues of the Epidermal Growth Factor-2 Domain of Factor IX by Alanine-scanning Mutagenesis

Abstract

This paper describes the consequences of alaninescanning mutagenesis on 28 positions of the second epidermal growth factor (EGF-2) domain of factor IX. We identified four positions of Gln 97 , Phe 98 , Tyr 115 , and Leu 117 that are critical for secretion of factor IX. Of the remaining mutations, 4 mutants (V86A, E113A, K122A, and S123A) are as active as wild-type factor IX (IXwt); 16 (D85A, K100A, N101A, D104A, N105A, R116A, E119A, T87A, I90A, K91A, R94A, E96A, S102A, K106A, T112A, and N120A) retain reduced but detectable activity, and 4 (N89A, N92A, G93A, and V107A) are nearly inert in the clotting assay. Both factor XIa and the factor VIIa-tissue factor complex effectively catalyzed the activation of these mutants except N89A. The mutant V107A failed to form the factor tenase complex with factor VIIIa because of a 35-fold increase in K d . The mutants N89A and N92A did not compete with factor IXwt for factor VIIIa binding, and G93A exhibited a 6-fold increase in K i values in the competitive binding assay. It appears that mutations at these positions have significantly affected the interaction between factor IX and factor VIIIa, although other mutations had little effect on the binding of factor IX to factor VIIIa. Mutations in two regions, Thr 87 -Gly 93 and Asn 101 -Val 107 , significantly increased the K m value of factor IXa (2-10-fold) in cleavage of factor X in the absence of factor VIIIa. In the presence of factor VIIIa, the catalytic efficiency of each mutant toward factor X paralleled its clotting activity. Briefly, we propose two relatively distinctive functions of factor IX for two adjacent regions in the EGF-2 domain; the first loop region (residues 89 -94) is involved with the binding of its cofactor, factor VIIIa, and the third loop with connected ␤-sheets (residues 102-108) is involved in the proper binding to the substrate, factor X.

The Factor IX γ-Carboxyglutamic Acid (Gla) Domain Is Involved in Interactions between Factor IX and Factor XIa

“…However, all of these mutations interfere with other activities of FIX in addition to activation by factor XIa. A rFIX protein containing the factor VII EGF1 domain is activated normally by factor XIa (53,54), whereas a study employing alanine scanning mutagenesis on portions of the EGF2 domain identified only a single amino acid (position 89) that appeared to be necessary for normal activation by factor XIa (55).…”

Identification of Residues Asn89, Ile90, and Val107 of the Factor IXa Second Epidermal Growth Factor Domain That Are Essential for the Assembly of the Factor X-activating Complex on Activated Platelets

“…Coagulation Assays-Activated partial thromboplastin time assays for the seven "candidate" alanine mutants were previously reported (8) and are listed in Table IV, which confirms the physiological function of the interrupted surface binding property of the FIX mutants in mediating clotting. The three mutants N89A (0.5%), I90A (7%), and V107A (0.5%) exhibited the lowest clotting activities.…”

“…Earlier time-course solution-phase FIX activation results have shown that FXIa activates all the seven FIX alanine mutants at normal rates compared with native FIX (8), and each of the FIX proteins was fully activated by FXIa in solution. Gelcode Blue-stained gels of SDS-PAGE displayed complete disappearance of the zymogen band (ϳ70 kDa) and appearance of heavy (ϳ28 kDa) and light chains (ϳ18 kDa) of FIXa for all the recombinant FIXa proteins (data not shown).…”

“…In Vitro Mutagenesis and Mutant FIX Expression-Alanine point mutations were generated by using the PCR-based method, FIX mutant proteins were expressed in HEK293 cells, and the highest expressing clones were identified as previously described (8). All wild type and mutant proteins were expressed at similar levels.…”

“…Occupancy of these binding sites is closely correlated with optimal rate enhancements of FX activation (Ͼ2 ϫ 10 8 -fold) in the presence of FVIIIa, emphasizing the physiological significance of platelet-receptor-mediated coagulation complex assembly (2,6,7). Recently, however, alanine-scanning mutagenesis studies of residues within the EGF2 domain of FIX have identified residues Asn 89 -Gly 93 that were implicated as critical for binding of FVIIIa (8). Because it is highly unlikely that the same subdomain of the FIXa EGF2 domain could be important for interactions with both platelet receptors and with FVIIIa, we have conducted the present studies, which are designed to identify specific amino acids essential either for binding to activated platelet receptors or to the cofactor, FVIIIa.…”