Figure 1.

Introduction of rat ACE2 residues into the human ACE2 catalytic domain interferes with S-protein association. (A) HEK293T cells were transfected with plasmids encoding human or rat ACE2, or chimeras of these receptors in which residues 1–600, corresponding to the ACE2 catalytic domain, were exchanged. Transfected cells were metabolically labeled with [³⁵S]cysteine and [³⁵S]methionine, and lysed. Lysates were immunoprecipitated with Protein A–Sepharose together with either the S1 domain of the SARS-CoV (TOR2) S protein fused to the Fc domain of human IgG1 (S1-Ig), or with an antibody (1D4) recognizing a tag present at the carboxy-terminus of each of the ACE2 variants, and analyzed by SDS–PAGE. (B) HEK293T cells were transfected with plasmids encoding human ACE2, rat ACE2, or human ACE2 variants in which residues corresponding to those of rat ACE2 were introduced at the indicated position. Transfected cells were analyzed as in (A) and precipitated ACE2 was quantified by phosphorimaging. Values indicate the ratio of protein precipitated by S1-Ig to that precipitated by 1D4. Error bars indicate the range of two or more experiments.