The IgA Isotype of Anti-β2 Glycoprotein I Antibodies Recognizes Epitopes in Domains 3, 4, and 5 That Are Located in a Lateral Zone of the Molecule (L-Shaped)

Abstract

Background: Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with presence of anti-phospholipid antibodies (aPL). The APS classification criteria only consider the aPL of IgG/IgM isotype, however testing of aPL of IgA isotype is recommended when APS is suspected and consensus aPL are negative. IgA anti-βeta-2 glycoprotein-I (B2GP1) has been clearly related with occurrence of thrombotic events. Antibodies anti-B2GP1 of IgG/M isotypes recognize an epitope in Domain 1 (R39-G43), the epitopes that recognize IgA anti-B2GP1 antibodies are not well-identified. Aim: To determine the zones of B2GP1 recognized by antibodies of IgA isotype from patients with APS symptomatology and positive for IgA anti-B2GP1. Methods: IgA antibodies to Domain-1(D1) and Domain-4/5(D4/5) of B2GP1 (ELISA) and epitope mapping on oligopeptide arrays of B2GP1 were evaluated in sera from a group of 93 patients with at least one thrombotic and with isolated positivity for IgA anti-B2GP1 antibodies (negative for other aPL). Results: A total of 47 patients (50.5%) were positive for anti-D4/5 and 23(25%) were positive for anti-D1. When peptide arrays were analyzed, three zones of B2GP1 reactivity were identified for more than 50% of patients. The center of these zones corresponds to amino acids 140(D3), 204(D4), and 264(D5). The peptides recognized on D3 and D4 contain amino acid sequences sharing high homology with proteins of microorganism that were previously related with a possible APS infectious etiology. In the three-dimensional structure of B2GP1, the three peptides, as the R39-G43 epitope, are located on the right side of the molecule (L-shape). The left side (J-shape) does not bind the antibodies. Conclusions: Patients with thrombotic APS clinical-criteria, and isolated IgA anti-B2GP1 positivity appear to preferentially bind, not to the D1 or D4/5 domains of B2GP1, but rather to three sites in D3, D4, and D5. The sites on D3 and D4 were previously described as the target identified by human monoclonal antibodies derived from patients that were capable of inducing APS in animal models. The localization of these epitopes opens a new route to explore to increase understanding of the patholophysiology of the APS and to propose new alternatives and therapeutic targets.

Keywords: B2GP1; antiphospholipid antibodies; antiphospholipid syndrome; epitope mapping; graft thrombosis; kidney transplant; peptide arrays.

Figure 1

Correlation of IgA levels of anti-B2GP1 vs. anti-D1 and anti-D4/5. (A) Correlation of the levels of IgA anti-D1 and IgA anti-B2GP1 in the 93 patients with thrombotic events. No correlation should be found (R = 0.183; p = 0.079). (B) Correlation of the levels of IgA anti-D4/5 and IgA anti-B2GP1 in the same patients. In the overall study, moderate correlation was found (R = 0.582; p < 0.001). The graph has been dividend into two zones: the shaded zone includes the patients in whom no correlation is observed (R = 0.08). When only the patients of the non-shaded area are analyzed, a high correlation is observed (R = 0.81; p < 0.001). The patients with renal failure are in blue and those with normal kidney function in red.

Figure 2

Identification of immunodominant regions of B2GP1. Description of the degree of antigenicity of the 79 peptides of B2GP1 evaluated in the 93 patients with thrombotic events. On the Y axis the percentage of sera that are positive for each of the peptides is indicated. Three peptides with high antigenicity corresponding to numbers 34 (P34; aa: 133–147), 48 (P48; aa 189–203), and 64 (P68; aa 253–267) were detected. Likewise, three areas of accumulation of peptides with positivity (gray overlap) located around these peptides can be observed: ZONE 1 (peptides 33–35) zone 2 (peptides 46–52), and zone 3 (peptides 62–67). The peptides corresponding to each domain are marked in: Domain 1, Yellow; Domain 2, Pink; Domain 3, Green; Domain 4, Orange; Domain 5, Dark Blue; Interlinking regions corresponding to two domains, cyan.

Figure 3

Immunodominant regions in the subgroup of patients D4/5 negative and in control group. (A) Description of the degree of antigenicity of the 79 peptides in the 27 patients with chronic renal disease and low values of anti-D4/5 antibodies. The four areas with accumulation of positivity are shaded in gray. (B) Antigenicity of the 79 peptides in the reference group of 18 asymptomatic carriers of IgA anti-β2GP1 (with other aPL negative). Response in the control group is more heterogeneous, however the three main antigenic zones can be identified. The peptides corresponding to each domain are marked in: Domain 1, Yellow; Domain 2, Pink; Domain 3, Green; Domain 4, Orange; Domain 5, Dark Blue; Interlinking regions corresponding to two domains, cyan.

Figure 4

Visualization of the 3D structure of B2GP1 from the sides resembling the letter “L” and the letter “J.” (A) “L side” of B2GP1. Three peptides of high antigenicity (P34, P48, and P64) are indicated in orange. The R39-R43 epitope is indicated in red. (B) Visualization of the four previous epitopes in the “J side” of the molecule. Only a part of the P48 corresponding to the angle or “elbow” of the molecule is clearly observed. (C) Visualization of the epitopes of high antigenicity in 4 intermediate steps of the rotation process from the form in L to the form in J. In this process, it can be verified that the part of the P64 observed in the J face corresponds to a group of amino acids that protrude and that are really located in the L face.

Figure 5

Localization of the four immunodominant zones in the structure of B2GP1. Visualization of the amino acids that correspond to each zone (marked in orange). (A) Observing the L face. (B) Observing the J face.