Reply to Rubinstein: Is Lgr4 essential for VSV- and VSV-G-pseudotyped lentiviral vector entry to cells? [Letters to the Editor]

This is a response to a letter by Rubinstein (1).In response to the concerns delineated in a letter by Menachem Rubinstein (1), we found that protein truncation without an endogenous signal peptide is commonly performed in co-immunoprecipitation (2). Furthermore, there was no aggregation or precipitation of misfolded protein in our co-immunoprecipitation assay (Fig. 5B of Ref. 3), and the truncated LGR4-ECD (25–528 amino acids) has well-demonstrated bioactivity, as shown in our previous paper (4). Moreover, the data from our pull-down (Fig. 5C) and Biacore assays (Fig. 5, D and E) confirmed that a strong interaction exists between Lgr4 and intact VSV particles (3).On the question of binding affinity, according to the affinity model, fast estimation of KD is reasonable only when the binding is saturated. In our case, it was hard to reach saturation for every concentration curve. Thus, as in other studies (see, for example, Ref. 5), the kinetics model was the only choice. Upon kinetic regression, the results showed that Kon = 15,450 m−1 s−1, Koff = 0.001067 s−1, and KD = Kd/Ka = 69.05 nm.Finally, the IC50 for LDLR-ECD cited by Dr. Rubinstein refers to sLDLR-mediated inhibition of vesicular stomatitis virus (VSV)–induced cytopathic effects in human WISH cells (Fig. 1A of Ref. 6). In contrast, we detected inhibition of VSV replication by LGR4-ECD in mouse MEF cells by quantitative PCR (Fig. 6A of Ref. 3). It is highly problematic to compare IC50 values determined using different assays, different cells, different expression systems, and different proteins.Taken together, we...

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