Abstract
Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that binds multiple distinct ligands, including blood coagulation factor VIII (FVIII). FVIII is a heterodimeric multidomain protein that consists of a heavy chain (domains A1, a1, A2, a2, and B) and a light chain (domains a3, A3, C1, and C2). Both chains contribute to high-affinity interaction with LRP. One LRP-interactive region has previously been located in the C2 domain, but its affinity is low in comparison with that of the entire FVIII light chain. We now have compared a variety of FVIII light chain derivatives with the light chain of its homolog FVa for LRP binding. In surface plasmon resonance studies employing LRP cluster II, the FVa and FVIII light chains proved different in that only FVIII displayed high-affinity binding. Because the FVIII a3-A3-C1 fragment was effective in associating with LRP, this region was explored for structural elements that are exposed but not conserved in FV. Competition studies using synthetic peptides suggested that LRP binding involves the FVIII-specific region Lys(1804)-Ala(1834) in the A3 domain. In line with this observation, LRP binding was inhibited by a recombinant antibody fragment that specifically binds to the FVIII sequence Glu(1811)-Lys(1818). The role of this sequence in LRP binding was further tested using a FVIII/FV chimera in which sequence Glu(1811)-Lys(1818) was replaced with the corresponding sequence of FV. Although this chimera still displayed residual binding to LRP cluster II, its affinity was reduced. This suggests that multiple sites in FVIII contribute to high-affinity LRP binding, one of which is the FVIII A3 domain region Glu(1811)-Lys(1818). This suggests that LRP binding to the FVIII A3 domain involves the same structural elements that also contribute to the assembly of FVIII with FIXa.
Highlights
Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that binds multiple distinct ligands, including blood coagulation factor VIII (FVIII)
Interaction between LRP and FVIII Light Chain Fragments— We have previously shown that the isolated FVIII C2 domain (i.e. Ser2173–Tyr2332) associates with LRP less effectively than the intact FVIII light chain (18)
We explored the possibility that additional sites in the FVIII light chain contribute to LRP binding
Summary
Materials—CNBr-Sepharose 4B was from Amersham Biosciences (Uppsala, Sweden). Microtiter plates (Maxisorp), cell culture flasks, Opti-MEM I medium, penicillin, and streptomycin were from Invitrogen (Breda, The Netherlands). After three rapid washes (Ͻ5 s each) with 150 mM NaCl, 5 mM CaCl2, 0.1% (v/v) Tween 20, and 50 mM Tris (pH 7.4), bound ligand was detected by incubation with peroxidase-conjugated monoclonal antibody CLB-CAg12 in the same buffer for 15 min at 37 °C During this latter incubation period, one would expect that the FVIII light chain would completely dissociate from the immobilized LRP clusters. ScFv KM36 (100 nM), scFv KM41 (40 nM), or LRP cluster II (25–125 nM) was passed over separate channels with the immobilized FVIII-(1811– 1818)/FV or recombinant wild-type FVIII light chain, respectively, and one control channel (scFv EL14-coated) in 150 mM NaCl, 2 mM CaCl2, 0.005% (v/v) Tween 20, and 20 mM Hepes (pH 7.4) for 2 min at a flow rate of 20 l/min at 25 °C. NM) or the scFv EL14-captured FVIII light chain (Kd ϭ 58 Ϯ 7 nM) was similar upon SPR analysis
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