Glycoproteomic identification of galectin-3 and -8 ligands in bronchoalveolar lavage of mild asthmatics and healthy subjects

BackgroundGalectins, a family of small carbohydrate binding proteins, have been implicated in regulation of inflammatory reactions, including asthma and fibrosis in the lungs. Galectins are found in cells of the airways and in airway secretions, but their glycoprotein ligands there have only been studied to a very limited extent.MethodsBronchoalveolar lavage (BAL) fluid from mild asthmatics and healthy volunteers were fractionated by affinity chromatography on the immobilized galectins. Total (10–30 μg) and galectin bound (~ 1–10 μg) protein fractions were identified, quantified and compared using shot-gun proteomics and spectral counts.ResultsAbout 175 proteins were identified in unfractionated BAL-fluid, and about 100 bound galectin-3 and 60 bound galectin-8. These included plasma glycoproteins, and typical airway proteins such as SP-A2, PIGR and SP-B. The concentration of galectin-binding proteins was 100–300 times higher than the concentration of galectins in BAL.ConclusionThe low relative concentration of galectins in BAL makes it likely that functional interactions with glycoproteins occur at sites rich in galectin, such as cells of the airways, rather than the extracellular fluid itself. The profile of galectin bound proteins differed between samples from asthma patients and healthy subjects and correlated with the presence of fibroblasts or eosinophils. This included appearance of a specific galectin-8-binding glycoform of haptoglobin, previously shown to be increased in serum in other inflammatory conditions.General significanceIt is technically feasible to identify galectin-binding glycoproteins in low concentration patient samples such as BAL-fluid, to generate biomedically interesting results. This article is part of a Special Issue entitled Glycoproteomics.

► BAL-fluid proteins were analyzed by mass spectrometry of a few microgram sample.
► Total (175) and proteins bound by galectin-3 (100) and -8 (60) were identified.
► The concentration of galectins in BAL-fluid was ~ 40 ng/ml or less.
► The summed concentration of galectin-binding proteins was > 100 times higher.
► Immunohistochemistry showed the two galectins in different cell types.