Localization of the low-density lipoprotein receptor-related protein regions involved in binding to the A2 domain of coagulation factor VIII

Abstract

SummaryCatabolism of coagulation factorVIII (FVIII) is mediated by lowdensity lipoprotein receptor-related protein (LRP). The ligandbinding sites of LRP are formed by complement-type repeats (CR), and CR clusters II and IV bind most of LRP ligands. FVIII contains two major LRP-binding sites located in the A2 and A3 domains. This study was aimed to identify specific complementtype repeats of LRP involved in interaction with the A2 site and to probe their functional importance in A2 catabolism. We generated individual LRP clusters II, III and IV, along with nine overlapping CR triplets encompassing clusters II and IV in a baculovirus expression system and studied their interaction with isolated A2. In surface plasmon resonance (SPR) assay, A2 bound to clusters II and IV with KDs 22 and 39 nM, respectively, and to the majority of CR triplets with affinities in the range of KDs 25–90 nM. Similar affinities were determined for A2 interaction with a panel of CR doublets overlapping cluster II (CR 3–4, 4–5, 5–6 6–7 and 7–8). These LRP fragments inhibited the binding of 125I-A2 to LRP in solid-phase assay,LRP-mediated internalization of 125I-A2 in cell culture and 125I-A2 clearance from the mouse circulation. Point mutations of critical A2 residues of the LRPbinding site resulted in differential reduction or abolishment of its binding to LRP fragments. We conclude that A2 interacts with LRP via multiple binding sites spanning CR 3–8 in cluster II and CR 23–29 in cluster IV, and the minimal A2-binding unit of LRP is formed by two adjacent CR.

Complement C1q Interacts With LRP1 Clusters II and IV Through a Site Close but Different From the Binding Site of Its C1r and C1s-Associated Proteases

“…The interaction of RAP with purified clusters II, III and IV was also investigated and gave K D values in the same nanomolar range of respectively 1.09 ± 0.31 nM (II), 1.06 ± 0.1 nM (III) and 1.28 ± 0.12 nM (IV) ( Figure 3, Table 1). These results are in agreement with previous studies (24)(25)(26) indicating that RAP interaction with LRP1 is strong and that the affinity of RAP for each individual cluster is in the same range. In the present study, RAP interaction allowed the validation of the functional integrity of the expressed LRP1 clusters.…”