Critical functions of L-selectin-mediated lymphocyte homing and recruitment
Lab/Group: Fukuda Lab (Burnham Institute)
Related Journal & Article Information
Journal: Nature Immunology
Article Title: Critical functions of N-glycans in L-selectin-mediated lymphocyte homing and recruitment
Introduction
To analyze glycan function, enzyme digestion is one of the easiest method. We successfully demonstrated the elimination of N-glycans, O-glycans and heparan sulfate on lymph node frozen section by N-glycanase (N-glycosidase F), O-sialoglycopeptidase, and heparitinases, respectively. N-glycanase treatment usually needs denaturing condition such as SDS treatment, but this can be substituted with acetone treatment of the frozen section.
Materials
Reagents
N-glycosidase F, Calbiochem.
O-sialoglycoprotein endopeptidase, Accurate Chemical and Scientific Corporation.
Heparitinase I and II and heparinase, Seikagaku.
Equipment
Procedure
Fixation
1. Fix lymph node frozen sections with acetone for 15 min at room temperature.
2. Wash the sections with PBS 3 times and treated with the following enzymes.
N-glycanase treatment
3. Incubate the fixed lymph node frozen sections with 100 mU/ml N-glycosidase F in 10 mM HEPES-NaOH, pH 7.4, 0.1% Triton X-100, 0.1 M 2-mercaptoethanol and complete protease inhibitor (Roche) at 37°C for 2 h. Proceed to 6.
O-sialoglycopeptidase treatment
4. Incubate the sections with 0.1 ~ 1 mg/ml O-sialoglycoprotein endopeptidase1 for in 10 mM HEPES-NaOH, pH 7.4 at 37°C for 2 h (ref. 1). Proceed to 6.
Heparitinases
5. Incubate the sections with 25 mU/ml Heparitinase I and II and heparinase in PBS containing 1 mg/ml BSA, 1 mM CaCl2 and the protease inhibitor at 37 °C for 2 h. Proceed to 6.
Washing
6. The digested sections were washed with PBS containing 0.1 mg/ml BSA at least 3 times and subjected to further staining.
Staining
7. Stanadard immunostaining technique using antibodies specific for appropriate glycans can be done to evaluate the digestions. Lectin staining including L/E/P-selectin-IgM staining may be also applicable2.
Troubleshooting
When the sections are treated with N-glycanase, weak L/E-selectin staining might persist. In that case, fix the section with 4% paraformaldehyde at room temperature for 15 min, denature proteins with 2% SDS and 5% 2-mercaptoethanol at 65 °C for 10 min. For O-sialoglycopeptidase treatment, it is very difficult to obtain the best results and trial and error may be necessary.
Critical Steps
The section should not be dried. Keep the sample in a moisture chamber
Anticipated Results
N-glycanse treatment removes all E/L-selectin-IgM binding in O-glycan deficient mice2. O-sialoglycopepeptidase digestion partially cleaves O-glycans but not always complete. Digestion with heparitinase I and II with heparinase eliminates all heparan sulfate as judged by staining with monoclonal antibody 10E4 (ref. 2).
References
1. Clark, R. A., Fuhlbrigge, R. C. & Springer, T. A. L-Selectin ligands that are O-glycoprotease resistant and distinct from MECA-79 antigen are sufficient for tethering and rolling of lymphocytes on human high endothelial venules. J Cell Biol 140, 721-731 (1998).
2. Mitoma, J., Bao, X., Petryniak, B., Schaerli, P., Gauguet, J. M., Yu, S.Y., Kawashima, H., Saito, H., Ohtsubo, K., Marth, J. D., Khoo, K. H., von Andrian, U. H., Lowe, J. B. and Fukuda, M. Critical functions of N-glycans in L-selectin-mediated lymphocyte homing and recruitment.
Nat Immunol. 8, 409-418. (2007).
Acknowledgements
supported by NIH grants CA71932 and CA48737
Keywords
L-selectin ligands,
lymphocyte homing,
high endothelial venules
N-glycans,
O-glycans,
heparan sulfate