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
. 2002 Apr;105(4):441-9.
doi: 10.1046/j.1365-2567.2002.01393.x.

Identification and characterization of L-selectin ligands in porcine lymphoid tissues

Adil I Khan  1 , Dorian O Haskard, Rajneesh Malhotra, R Clive Landis
Affiliations

Identification and characterization of L-selectin ligands in porcine lymphoid tissues

Adil I Khan et al. Immunology. 2002 Apr.

Abstract

A human L-selectin-ZZ fusion protein was used to screen porcine inguinal lymph nodes for the presence of monoclonal antibody (mAb) MECA 79-negative ligands. Fractionation of lymph node-conditioned medium by anion-exchange chromatography revealed two distinct L-selectin-binding fractions, one containing a MECA 79 non-reactive species and the second containing two MECA 79 reactive species of approximately 84 000 and 210 000 molecular weight. The MECA 79 non-reactive species exhibited Ca2+- and lectin-dependent binding to L-selectin-ZZ in a solid-phase capture enzyme-linked immunosorbent assay (ELISA), and this was specifically disrupted by the addition of EDTA, mannose-6-phosphate and the blocking anti-L-selectin mAb, DREG-56. Enzymatic characterization of the ligand by trypsin, O-sialoglycoprotease endopeptidase, heparinases I and III, or chondroitinase ABC lyase digestion indicated that L-selectin binding was predominantly dependent upon a chondroitin sulphate-modified glycoprotein determinant. Although Coomassie Blue staining of sodium dodecyl sulphate (SDS) polyacrylamide gels did not reveal a detectable protein species, carbohydrate-specific staining using GlycoTrack revealed a single, heavily glycosylated protein of high molecular weight (> 220 000). These studies have revealed the existence of a MECA 79 non-reactive, chondroitin sulphate glycosaminoglycan-modified ligand, termed csgp>220, which is secreted by peripheral lymph nodes and may play a role in leucocyte trafficking to the lymph node.

PubMed Disclaimer

Figures

Figure 1
Figure 1
L-selectin binding and MECA 79 reactivity of anion-exchange fractionated lymph node-conditioned medium. (a) Binding of anion-exchange fractions to L-selectin–ZZ in a solid-phase enzyme-linked immunosorbent assay (ELISA). Microtitre wells were coated with 100 µl of fraction (diluted 1 : 5 to lower the salt content) and binding was determined by the addition of L-selectin–ZZ and horseradish peroxidase (HRP)-conjugated swine anti-rabbit immunoglobulin G (IgG), followed by calorimetric determination at 490 nm. (b) Pooled fractions were precleared using protein A to remove IgG contaminants and dialysed into Tris assay buffer, before reanalysis by solid-phase ELISA. (c) Western blot analysis of pooled fractions A to D with monoclonal antibody (mAb) MECA 79. Pooled fractions (20 µl) were separated under non-reducing conditions on a 10% sodium dodecyl sulphate (SDS) polyacrylamide gel and transferred to nitrocellulose membranes for detection using MECA 79. Fraction D, which exhibited the greatest binding capacity for L-selectin–ZZ, showed no detectable staining with MECA 79, whereas fraction B, with the second highest L-selectin binding capacity, contained two MECA 79-reactive species migrating at 84 kDa and 210 kDa, respectively. Bar charts depict the mean ± standard deviation (SD) of triplicate determinations.
Figure 2
Figure 2
Specificity of L-selectin binding by MECA 79-negative ligand(s) in fraction D. Panel (a) depicts specific binding by fraction D to L-selectin–ZZ, but not to E-selectin–ZZ, in the presence and absence of 10 mm EDTA. Panels (b) and (c) depict the dose-dependent inhibition of L-selectin–ZZ binding in the presence of increasing concentrations of mannose-6-phosphate and anti-L-selectin monoclonal antibody (mAb) DREG 56, respectively. Data represent mean ± standard deviation (SD) (n = 3).
Figure 3
Figure 3
Biochemical characterization of the L-selectin ligand(s) in fraction D. Microtitre wells were coated with ligand from fraction D, and binding to L-selectin–ZZ was assessed in the presence and absence of enzymatic treatment, as follows. (a) Heparinases I and III exerted no effect on L-selectin binding, ruling out the involvement of heparan sulphate determinants. (b) Trypsin followed by reversal with soya-bean trypsin inhibitor (T, I) reduced L-selectin binding by 30% compared to untreated, inhibitor only (I), or simultaneous treatment with trypsin in the presence of inhibitor (T + I). (c) Arthrobacter ureafaciens sialidase treatment reduced L-selectin binding by 17%. (d) O-sialoglycoprotease endopeptidase (OSGP) treatment inhibited L-selectin reactivity by 23%. Data represent mean ± standard deviation (SD) (n = 3).
Figure 4
Figure 4
Inhibition of L-selectin binding by chondroitin ABC lyase. Fraction D was immobilized to microtitre wells (as described in the Materials and methods) and treated with chondroitin ABC lyase, at the concentrations indicated, for 18 hr at 37°. Chondroitin ABC lyase treatment caused a dose-dependent inhibition of L-selectin–ZZ binding, achieving maximal inhibition (82%) at ≥ 0·1 U/ml. Data represent mean ± standard deviation (SD) (n = 3).
Figure 5
Figure 5
Analysis of the L-selectin ligand by Glycotrack™. Fraction D was incubated overnight with L-selectin–ZZ agarose beads, after which bound material was eluted with 10 mm EDTA. The eluate was subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) under non-reducing conditions and transferred to nitrocellulose. Glycosylated oligosaccharide residues were identified using the Glycotrack carbohydrate detection kit (Oxford GlycoSystems, Abingdon, UK), which revealed a single high molecular weight species migrating at > 220 kDa.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Gallatin WM, Weissman IL, Butcher EC. A cell-surface molecule involved in organ-specific homing of lymphocytes. Nature. 1983;304:30–4. - PubMed
    1. Lasky LA, Singer MS, Yednock TA, et al. Cloning of a lymphocyte homing receptor reveals a lectin domain. Cell. 1989;56:1045–55. - PubMed
    1. Stoolman LM, Rosen SD. Possible role for cell-surface carbohydrate-binding molecules in lymphocyte recirculation. J Cell Biol. 1983;96:722–9. - PMC - PubMed
    1. Stoolman LM, Tenforde TS, Rosen SD. Phosphomannosyl receptors may participate in the adhesive interaction between lymphocytes and high endothelial venules. J Cell Biol. 1984;99:1535–40. - PMC - PubMed
    1. Imai Y, Singer MS, Fennie C, Lasky LA, Rosen SD. Identification of a carbohydrate-based endothelial ligand for a lymphocyte homing receptor. J Cell Biol. 1991;113:1213–21. - PMC - PubMed

Publication types

LinkOut - more resources