Characterization of SpsQ from Staphylococcus pseudintermedius as an affinity chromatography ligand for canine therapeutic antibodies

Abstract

Coagulase-positive Staphylococci express protein A, which binds to host antibodies, to evade the immune system. Taking advantage of its specific binding to antibodies, protein A from Staphylococcus aureus, which is called SpA, is commonly used as an affinity chromatography ligand for human therapeutic antibodies. However, among four canine IgG subclasses (A, B, C, and D), only IgG-B binds to SpA strongly and establishing an efficient and robust purification scheme for canine therapeutic antibodies whose IgG subclass is A, C, or D remains difficult and depends on finding a suitable substitute to SpA. S. pseudintermedius, a major coagulase-positive Staphylococci found in dogs, expresses spsQ gene which is orthologous to S. aureus spa. We hypothesized that to serve S. pseudintermedius to better adapt to the dog immune system, SpsQ would bind to canine IgGs stronger than SpA, making it a better affinity chromatography ligand for canine therapeutic antibodies. To characterize SpsQ, we first determined the spsQ nucleotide sequence from S. pseudintermedius isolates. Based on the identified sequence, we prepared recombinant proteins containing the immunoglobulin-binding domains of SpA (r-SpA) and SpsQ (r-SpsQ) and determined their binding capacity for each canine IgG subclass. The binding capacity of r-SpsQ for IgG-B was almost as high as that of r-SpA. Interestingly, while both r-SpsQ and r-SpA showed no binding to IgG-C, the binding capacity of r-SpsQ for IgG-A and IgG-D was significantly higher than that of r-SpA. Finally, we performed affinity chromatography using r-SpsQ- or r-SpA-immobilized resin and revealed that the recovery rates of IgG-A and IgG-D using r-SpsQ were significantly higher than those using r-SpA. Our findings indicate that SpsQ has a strong potential to be used as an affinity chromatography ligand for canine therapeutic antibodies of subclass A, B, and D.

Fig 1. Determination of the Nucleotide Sequence of spsQ from S. pseudintermedius Isolates.

(a) Alignment of SpsQ. Predicted Ig-BDs are indicated. (b) Schematics of the predicted functional motifs of SpsQ and SpA. (c) Alignment of each Ig-BD from SpsQ with domain C from SpA of Staphylococcus aureus subsp. aureus NCTC 8325 (GenBank accession number; CP000253.1).

Fig 2. Preparation of recombinant SpsQ and SpA.

The amino acid sequences of (a) recombinant SpsQ (r-SpsQ) and (b) recombinant SpA (r-SpA). Amino acid residues derived from the expression vector are underlined. The N-terminal 10 × his-tag sequence is shown by a red box. (c) SDS-PAGE of the purified recombinant proteins. Arrowheads indicate the specific protein bands with an expected molecular weight under reducing conditions.

Fig 3. Evaluation of the binding of r-SpsQ or r-SpA to Canine IgGs.

The comparison of the binding of r-SpsQ and r-SpA to canine (a) IgG-A, (b) IgG-B, (c) IgG-C, and (d) IgG-D, evaluated by ELISA. Briefly, canine IgGs were coated on the ELISA plates and biotinylated r-SpsQ or r-SpA was added onto the plate at various concentrations (horizontal axis). The binding of r-SpsQ or r-SpA was detected with HRP-conjugated Avidin and colorimetric substrate. The y-coordinate of each dot represents the average absorbance obtained from three independent experiments. Error bars represent the standard deviation. Tukey’s test was used for statistical comparison (*: p < 0.01).

Fig 4. Purification of canine IgGs using r-SpsQ and r-SpA.

(a) SDS-PAGE of the cell culture supernatant (SUP) and the recovered canine IgG-A, IgG-B, and IgG-D using r-SpsQ- or r-SpA-immobilized resin under reducing conditions. The recovery rate of (b) IgG-A, (c) IgG-B, and (d) IgG-D using r-SpsQ- or r-SpA-immobilized resin. The recovery rate is expressed as the recovered fraction (percentage) of the total amount of antibody loaded onto the resins (recovery rate % = amount (μg) of recovered antibody/loaded antibody ×100). Data points represent the average of three independent experiments. Error bars represent the standard deviation estimated from triplicates. A two-tailed t-test was used to compare groups.