Abstract
A novel galectin cDNA (galectin-14) was cloned from ovine eosinophil-rich leukocytes by low stringency reverse transcriptase-PCR and cDNA library screening. Data base searches indicate that this gene encodes a novel prototype galectin that contains one putative carbohydrate recognition domain and exhibits most identity to galectin-9/ecalectin, a potent eosinophil chemoattractant. The sugar binding properties of the recombinant molecule were confirmed by a hemagglutination assay and lactose inhibition. The mRNA and protein of galectin-14 are expressed at high levels in eosinophil-rich cell populations. Flow cytometry and cytospot staining demonstrate that the protein localizes to the cytoplasmic, but not the granular, compartment of eosinophils. In contrast, galectin-14 mRNA and protein were not detected in neutrophils, macrophages, or lymphocytes. Western blot analysis of bronchoalveolar lavage fluid indicates that galectin-14 is released from eosinophils into the lumen of the lungs after challenge with house dust mite allergen. The restricted expression of this novel galectin to eosinophils and its release into the lumen of the lung in a sheep asthma model indicates that it may play an important role in eosinophil function and allergic inflammation.
Highlights
The immune response of mammals to multicellular parasite infections and allergens is characterized by the recruitment of eosinophils [1, 2]
The expression of galectin-14 was up-regulated in the lung tissue of sensitized sheep challenged with house dust mite extract (HDM),1 and the protein was released into the bronchoalveolar lavage (BAL) fluid
Note that the various galectin-9 isoforms all give the same amino acid and nucleotide identity, because the putative galectin-14 protein finishes at the end of the NH2 terminal CRD, and does not cross the linker sequence that varies between these different isoforms
Summary
Collection of Mammary Lavage (MAL) Samples—To induce eosinophil migration into the mammary gland, mature non-lactating Merino ewes were primed every 2 weeks by intramammary infusions of 1 mg of solubilized house dust mite extract (HDM, Dermatophagoides pteronyssinus, Commonwealth Serum Laboratories Ltd., Melbourne, Victoria, Australia), rested for 3– 4 weeks and challenged with an intramammary infusion of 1 mg of solubilized HDM. MAL was collected 2 days postHDM challenge by infusion of sterile pyrogen-free saline (PFS, Baxter Healthcare Pty. Ltd., New South Wales, Australia) followed by milking of the gland as described previously [3, 4]. The. The sequences of adapters and primers used in low stringency or conventional RT-PCR are shown. Nucleotide substitutions introduced in primers G14 5ЈpGEX and G14 3ЈpGEX to alter codon usage to that preferred by E. coli are shown in boldface
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