Critical functions of L-selectin-mediated lymphocyte homing and recruitment

doi:10.1038/nprot.2007.282

Method Article

Critical functions of L-selectin-mediated lymphocyte homing and recruitment

https://doi.org/10.1038/nprot.2007.282

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Biochemistry

Computational biology and bioinformatics

Biological techniques

L-selectin ligands

lymphocyte homing

high endothelial venules

N-glycans

O-glycans

heparan sulfate

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.

N-glycosidase F, Calbiochem.

O-sialoglycoprotein endopeptidase, Accurate Chemical and Scientific Corporation.

Heparitinase I and II and heparinase, Seikagaku.

Fixation

Fix lymph node frozen sections with acetone for 15 min at room temperature.
Wash the sections with PBS 3 times and treated with the following enzymes.

N-glycanase treatment

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

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

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

The digested sections were washed with PBS containing 0.1 mg/ml BSA at least 3 times and subjected to further staining.

Staining

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.

5 hrs

The section should not be dried. Keep the sample in a moisture chamber

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.

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).

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).
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).

Supported by NIH grants CA71932 and CA48737

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Critical functions of L-selectin-mediated lymphocyte homing and recruitment

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Version 1

Introduction

Reagents

Procedure

Timing

Critical Steps

Troubleshooting

Anticipated Results

References

Acknowledgements

Associated Publications

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Critical functions of L-selectin-mediated lymphocyte homing and recruitment

Status:

Version 1

Introduction

Reagents

Procedure

Timing

Critical Steps

Troubleshooting

Anticipated Results

References

Acknowledgements

Associated Publications

Status:

Version 1

Privacy Policy

Terms of Service

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