Putative sequences on Ro60 three-dimensional structure accessible for 4-hydroxy-2-nonenal (HNE) modification compared to in vitro HNE modification of Ro60 sequences

Abstract

We have previously reported accelerated acquisition of new autoreactivity upon immunization with 4-hydroxy-2-nonenal (HNE)-modified Ro60, as well as differential induction of lupus or Sjögren's syndrome by immunization with Ro60 containing varying amounts of HNE. Since the number of HNE molecules on Ro60 appears to be important, we hypothesized that specific sequences on Ro60 are targets for HNE-modification. Using surface plasmon resonance (SPR) we have also shown intramolecular protein-protein interaction between Ro60 and Ro multiple antigenic peptides (MAPs). We also hypothesized that intramolecular protein-protein interaction would be abolished by HNE-modification. To test this hypotheses we investigated (a) the epitopes of Ro60, using 19 Ro MAPs in an in vitro assay (involving HNE-modification of MAPs following immobilization on ELISA plates) to identify targets of HNE modification on Ro60 and (b) the protein-protein interaction between unmodified Ro60 MAPs, immobilized on the sensor surface of BIAcore, and unmodified Ro60 or HNE-modified Ro60 using SPR. New data obtained with SPR strengthens our earlier observation that immunization with HNE-Ro60 induces a stronger response. Unmodified Ro60 bound to several Ro60 MAPs through protein-protein interaction analyzed using SPR. This interaction was totally abrogated using HNE-modified Ro60 suggesting that sequences on Ro had become modified with HNE. When 19 Ro60 MAPs were modified in vitro with HNE, it was found that 10/19 MAPs significantly bound HNE covalently (p<0.001 compared to MAPs binding HNE poorly). The amino acid sequences 126-137, 166-272 and 401-495 on Ro60 were strongly HNE modified. Using computational model system based on the recently published crystal structure for Ro60 enabled us to identify regions on the Ro60 molecule represented by the HNE-modified Ro MAPs, which are part of the exposed tertiary structure of the Ro60 protein.

Figure 1:

SPR analysis of rabbit anti-Ro60 or anti-HNE Ro60 interacting with Ro-MAP-310 coupled to a sensor surfaces. Rabbits were immunized with either Ro60 or HNE-modified Ro60. Top panel- Anti-Ro60 binding to Ro-MAP-310. Bottom panel- Anti-HNE-Ro60 interacting with Ro-MAP-310.

Figure 2:

SPR analysis of rabbit anti-Ro60 or anti-HNE Ro60 interacting with Ro-MAP-190 and Ro-MAP-401 coupled to separate sensor surfaces. Rabbits were immunized with either Ro60 or HNE-modified Ro60. A- Anti-Ro60 binding to Ro-MAP-190. B- Anti-HNE-Ro interacting with Ro-MAP-190. C: Anti-Ro60 interacting with Ro-MAP-401. D: Anti-HNE-Ro interacting with Ro-MAP-401.

Figure 3:

SPR analysis of rabbit anti-Ro60 or anti-HNE Ro60 interacting with Ro-MAP-449 and Ro-MAP-482 coupled to separate sensor surfaces. Rabbits were immunized with either Ro60 or HNE-modified Ro60. A - Anti-Ro60 binding to Ro-MAP-449. B- Anti-HNE-Ro interacting with Ro-MAP-449. C: Anti-Ro60 interacting with Ro-MAP-482. D: Anti-HNE-Ro60 interacting with Ro-MAP-482.

Figure 4:

SPR analysis of the protein–protein interaction of Ro60 or HNE-Ro60 with Ro-MAP-331 and Ro-MAP-482 coupled to sensor surface. A- HNE Ro60 interacting with Ro-MAP-331. B- Ro60 interacting with Ro-MAP-331. C- HNE Ro60 interacting with Ro-MAP-482. D- Ro60 interacting with Ro-MAP-482.

Figure 5:

Binding of anti-HNE to unmodified and HNE-modified Ro60 MAPs. MAPs that were modified with HNE (post immobilization) or not modified with HNE were tested for their ability to bind anti- HNE antibodies. The MAPs are designated by their numbers assigned as given in Table 1. Values are means ± S.D. for four determinations.

Figure 6:

Crystal structure of the 60 kD Ro molecule [PDB ID: 1YVP] in worm representation. The colored portions with corresponding numbers in each case represents the sequence of each individual peptide given in Table 1. Ro60 MAPs 4, 7, 9, 10, 11, 12, 13, 19, 20, 21 were significantly modified by HNE compared to Ro60 MAPs 2, 3, 5, 6, 8, 14, 15, 16 and 17. This figure was made by the program MOLMOL (Koradi et al, 1996).