Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Feb;44(2):238-43.
doi: 10.1165/rcmb.2010-0007OC. Epub 2010 Apr 15.

Characterization of expression of glycan ligands for Siglec-F in normal mouse lungs

Affiliations

Characterization of expression of glycan ligands for Siglec-F in normal mouse lungs

Jin P Guo et al. Am J Respir Cell Mol Biol. 2011 Feb.

Abstract

Sialic acid-binding immunoglobulin-like lectin (Siglec)-F, an inhibitory receptor on mouse eosinophils, preferentially recognizes the glycan ligand 6'-sulfated sialyl Lewis X, but little is known about the requirements for its lung expression. RT-PCR and immunohistochemistry were used to detect and localize the sulfotransferase keratin sulfate galactose 6-O sulfotransferase (KSGal6ST, also known as carbohydrate sulfotransferase 1; gene name, Chst1) that is putatively required for 6'-sulfated Sialyl Lewis X synthesis. RT-PCR detected the greatest constitutive expression of Chst1 in lung, liver, and spleen tissue. Immunohistochemistry localized the expression of KSGal6ST in lung tissue primarily to airway epithelium. Siglec-F-Ig fusion protein selectively bound in a similar pattern, and was unaffected in lung tissue treated with methanol or deficient in Type 2 α2,3 sialyltransferase (St3gal2), but was eliminated by proteinase K or sialidase, and was absent in tissue deficient in the Type 3 α2,3 sialyltransferase (St3gal3). Binding of the Siglec-F-Ig fusion protein was similar in pattern to, and completely blocked by, a plant lectin recognizing α2,3-linked sialic acid. Thus, α2,3-linked sialic acid-containing glycoprotein Siglec-F ligands and the enzymes required for their synthesis are constitutively expressed in murine lungs, especially by airway epithelium. St3gal3, but not St3gal2, is required for constitutive Siglec-F ligand synthesis. The survival of eosinophils entering the lung may be shortened by encountering these Siglec-F sialoside ligands.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Constitutive expression of keratin sulfate galactose 6-O-sulfotransferase (KSGal6ST) gene Chst1 was detected using RT-PCR in wild-type C57BL/6 mouse tissues. L, lung; H, heart; Li, liver; K, kidney; Sk, skin; I, intestine; Spl, spleen, with DNA ladder also shown at left. Results from one of two experiments with similar findings are shown. The amplified KSGal6ST fragment consisted of 553 base pairs, whereas the β-actin fragment consisted of 192 base pairs.
Figure 2.
Figure 2.
Localization of KSGal6ST protein expression in normal mouse lung. (A) Two separate whole mouse lung extracts were run on gels in paired lanes (lanes 1–4) and probed with two different polyclonal antibodies recognizing KSGal6ST (Gel #1, probed with the rabbit antibody, lanes 1 and 2; Gel #2, probed with the sheep antibody, lanes 3 and 4). A single band occurred at approximately 45 kD. (B) Immunohistochemical localization of KSGal6ST protein in tissue sections of normal mouse lungs, using rabbit polyclonal antibody. Epithelial staining is particularly pronounced, but staining is also evident in some alveolar structural cells. Staining is blocked by preincubation with the KSGal6ST peptide used to create the polyclonal antibody. Results are shown from one of four experiments with identical findings. Original magnification, ×100. Original magnification, ×250 for insets.
Figure 3.
Figure 3.
Binding characteristics of control human IgG (Omalizumab), Siglec–F–Ig fusion protein, and Siglec-10–Ig fusion protein in normal murine lungs without (A) or with (B) sialidase treatment. Staining is predominantly epithelial, with some alveolar cell labeling, and is eliminated by treatment with sialidase. Results are shown from one of three experiments with identical findings. Original magnification, ×100. Original magnification, ×200 for inset in A for Siglec-F–Ig binding, to provide greater details of cellular staining patterns.
Figure 4.
Figure 4.
Binding of Siglec-F–Ig fusion protein to normal mouse lung without (A) or with (B) proteinase K treatment. Staining in both models was completely eliminated by protease treatment. Results are shown from one of three experiments with similar results. Original magnification, ×100.
Figure 5.
Figure 5.
Contributions of Type 2 α2,3 sialyltransferase (St3gal2) and Type 3 α2,3 sialyltransferase (St3gal3) to the binding of Siglec-F–Ig fusion protein in murine lungs. C57BL/6 wild-type, St3gal2−/−, and St3gal3−/− murine lungs were examined as in Figure 4. (A) IgG control staining. (B) Siglec-F–Ig fusion protein staining. Staining intensity and pattern in wild-type and St3gal2−/− mice were identical, whereas St3gal3−/− murine lungs were nearly devoid of staining. Results are shown from one of three experiments with similar results. Original magnification, ×100.
Figure 6.
Figure 6.
(A) Binding characteristics of Maackia amurensis (MAA) lectin and Sambucus nigra (SNA) lectin to normal murine lungs with or without sialidase treatment, as indicated. Staining patterns for MAA, but not SNA, are similar to Siglec-F–Ig fusion protein. Both MAA and SNA binding was completely eliminated with sialidase treatment. Results are shown from one of four experiments with similar findings. (B) Effect of pretreatment with MAA lectin or SNA lectin on binding of Siglec-F–Ig fusion protein (Siglec-F-Fc). Preincubation with MAA, but not SNA, blocked binding of the Siglec-F–Ig fusion protein. Results are shown from one of two experiments with identical findings. Original magnification, ×100.

Similar articles

Cited by

References

    1. Kikly KK, Bochner BS, Freeman S, Tan KB, Gallagher KT, D'Alessio K, Holmes SD, Abrahamson J, Hopson CB, Fischer EI, et al. Identification of SAF-2, a novel Siglec expressed on eosinophils, mast cells and basophils. J Allergy Clin Immunol 2000;105:1093–1100. - PubMed
    1. Floyd H, Ni J, Cornish AL, Zeng Z, Liu D, Carter KC, Steel J, Crocker PR. Siglec-8: a novel eosinophil-specific member of the immunoglobulin superfamily. J Biol Chem 2000;275:861–866. - PubMed
    1. Varki A, Angata T. Siglecs: the major sub-family of I-type lectins. Glycobiology 2006;16:1R–27R. - PubMed
    1. Crocker PR, Paulson JC, Varki A. Siglecs and their roles in the immune system. Nat Rev Immunol 2007;7:255–266. - PubMed
    1. von Gunten S, Bochner BS. Basic and clinical immunology of Siglecs. Ann N Y Acad Sci 2008;1143:61–82. - PMC - PubMed

Publication types

MeSH terms