Antigenic changes in human albumin caused by reactivity with the occupational allergen diphenylmethane diisocyanate

Abstract

Diphenylmethane diisocyanate (MDI), the chemical commonly used as a cross-linking agent in commercial polyurethane production, is a well-recognized cause of asthma. Reaction products between MDI and "self" proteins are hypothesized to act as antigens capable of inducing airway inflammation and asthma; however, such MDI antigens remain incompletely understood. We used a variety of analytical methods to characterize the range of MDI-albumin reaction products that form under physiological conditions. Sites of MDI conjugation on antigenic MDI-albumin products, as defined by serum immunoglobulin G (IgG) from MDI-exposed workers, were determined by high-performance liquid chromatography (HPLC) followed by tandem mass spectrometry (MS/MS). The data identified 14 MDI conjugation sites (12 lysines and 2 asparagines) on human albumin and highlight reaction specificity for the second lysine in dilysine (KK) motifs, and this may be a common characteristic of "immune-sensitizing" chemicals. Several of the MDI conjugation sites are not conserved in albumin from other species, and this may suggest species differences in epitope specificity for self protein (albumin)-isocyanate conjugates. The study also describes new applications of contemporary proteomic methodology for characterizing and standardizing MDI-albumin conjugates destined for use in clinical research.

Fig. 1

MDI reactivity with human albumin changes its conformation and charge. Reaction products between MDI and human albumin were analyzed by electrophoresis under nonreducing/SDS conditions (A), reducing/SDS conditions (B), or native conditions (C). The relative amount of the reactants (MDI/albumin) at the beginning of the reactions was varied from 0 to 1000 μg MDI/mg albumin as indicated.

Fig. 2

Variability in ELISA detection of specific IgG by MDI–albumin conjugates. MDI–albumin conjugates generated with different amounts of MDI, ranging from 0 to 1000 μg MDI/mg albumin, were used (as antigens) to coat microtiter plates in an ELISA for human IgG binding using serum samples from 10 MDI-exposed subjects and 2 representative unexposed individuals. All serum samples were diluted 1:200. The y axis reflects the amount of specific IgG binding. All samples were tested in duplicate, and the results shown are representative of three independent experiments.

Fig. 3

Separation of MDI-conjugated (albumin) peptides by HPLC. Control (red) or MDI-conjugated (blue) albumin was trypsin digested and fractionated by HPLC as described in Materials and methods. The absorbance of the column elution at 245 nm (y axis) is plotted over time in minutes (x axis). Note different scales for the MDI–albumin samples (left axis) versus the control albumin samples (right axis). Data shown are representative of four independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Comparison of human versus other species' albumin and mapping of MDI conjugation sites. (A) Primary amino acid sequence of the mature albumin protein from human, bovine, and murine species (GenBank Accession Nos. ABS29264.1, AAA51411.1, and AAH49971.1 respectively) aligned using the constraint-based multiple alignment tool from the National Center for Biotechnology Information. MDI conjugation sites in human albumin are depicted as red letters, and those not conserved in murine and bovine albumin are circled. (B) Unique MDI conjugation sites in human albumin. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Localization of MDI conjugation sites on human albumin. The sites of MDI conjugation identified by HPLC–MS/MS are highlighted on a three-dimensional representation of human albumin (1UOR) obtained from the protein/molecular modeling database and are displayed using the Cn3D program from the National Center for Biotechnology Information.

Fig. 6

Specificity of the human immune response to MDI: Dependence on human albumin. MDI–albumin conjugates were prepared with albumin from different species as described in Materials and methods and were used as antigens in an ELISA for specific human IgG binding (y axis). Representative experiments performed with serum from 3 MDI-exposed subjects and 1 unexposed control subject are shown.