CD-31

​CD-31 (cluster of differentiation 31) is a transmembrane protein encoded by the PECAM1 (platelet endothelial cell adhesion molecule) gene. CD-31 has essential roles in platelet biology, signal transduction, and leukocyte and endothelial cell biology.1 

Overview​​

​​

​​Protein function, expressions, and isoforms

Protein function

  • CD-31 is a cell adhesion molecule that is required for leukocyte transendothelial migration (TEM) under most inflammatory conditions. 2 3
  • Heterophilic interaction with CD177 plays a role in the transendothelial migration of neutrophils. 3 
  • ​Homophilic ligation of PECAM1 prevents macrophage-mediated phagocytosis of neighboring viable leukocytes by transmitting a detachment signal. 4
  • Promotes macrophage-mediated phagocytosis of apoptotic leukocytes by tethering them to the phagocytic cells; PECAM1-mediated detachment signal appears to be disabled in apoptotic leukocytes. 4
  • Modulates bradykinin receptor BDKRB2 activation. Regulates bradykinin- and hyperosmotic shock-induced ERK1/2 activation in endothelial cells. 5

​Expression

  • Expressed on platelets, leukocytes, neutrophils 3, human umbilical vein endothelial cells (HUVECs). 2 3
  • CD-31 is primarily concentrated at the borders between endothelial cells. 6 7
  • Isoform long predominates in all tissues examined. 8

​​Isoforms

  • Human: Isoform 1-6: 79-83kD (predicted)
  • Mouse: Isoform 1-4: 69.8-81.3kD (predicted)

​​Applications

CD-31 can be a challenging target to work with in some applications.

Western blot

CD31 is a transmembrane protein 3, 7 that requires special treatment of samples in western blot. It contains many post-transcriptional modifications, including glycosylations and phosphorylations 2, 7, 9,10,11,12, making the actual band size around 130kD, different from the predicted 79-83kD.




Sample preparation

Add adequate protease inhibitors (or phosphatase inhibitors for proteins modified by phosphorylation) to avoid target protein degradation.


Keep samples on ice during the whole WB process.


Perform a Bradford assay, a Lowry assay, or a bicinchoninic acid (BCA) assay to determine the protein concentration.

Electrophoresis

For large proteins (the MW of target protein >100 kDa), be sure to run samples in 8% or lower separating gel.


Load 20-50μg total protein per lane.

Transferring

It is preferred to add SDS to a final concentration of 0.1% in the transfer buffer for large proteins.


Use Ponceau S staining to determine if the transfer is successful.

Maximizing signal

Treat samples with PNGase F or phosphatase to confirm the specificity of bands if
necessary.

Controls

Positive: 
HUVEC and Jurkat cell lysates (ab7899).
Human spleen and kidney tissue lysate.
Negative:
NIH/3T3 whole cell lysate (ab7179)

​​Immunohistochemistry (IHC)

CD31 is widely expressed on platelets and leukocytes and is primarily concentrated at the
borders between endothelial cells. 6, 7 It can be used to help identify blood vessels and endothelial cells. Pathologists use it to identify vascular origin of tumors, but nodal sinuses may show signal as well.13 CD31 is also used to identify vascular invasion 14, 15 and assess the micro-vessel density of tumors. 16



Sample preparation

The ideal fixation time will depend on the size of the tissue block and the type of tissue, but fixation between 18-24h is suitable for most samples. 

Antibody incubation

It is recommended to optimize antibody dilution in preliminary experiments according to datasheets.

Controls

Positive: 

Human tonsil tissue

​

​​Immunocytochemistry/Immunofluorescence (ICC/IF)

CD31 expresses in endothelial cells, B cells, platelets, macrophages, monocytes, NK cells, T cells. 6, 7 It is one of the biomarkers of endothelial cells. It is widely used to confirm the cell type and co-localization of proteins of interest in immunocytochemistry application.



Sample preparation

It is recommended to fix cells in 4% PFA for 20 minutes at room temperature.


Do not over-fix your samples, which will reduce signal

Permeabilization

It is recommended to incubate cells with 0.1% Triton-X for 5 min to detect nuclear antigen.

Antibody incubation

Use 0.3M glycine to quench autofluorescence caused by aldehydes.

Controls

Positive: 

HUVEC cells

Find full information on working with CD-31:

Find full information on working with CD-31:
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​​References

  1. Lertkiatmongkol, P., Liao, D., Mei, H., Hu, Y., Newman, P.J. Endothelial functions of platelet/endothelial cell adhesion molecule-1 (CD31). Curr Opin Hematol. 23, 253-259 (2016).  
  2. Dasgupta, B., Dufour, E., Mamdouh, Z., Muller, W.A. A novel and critical role for tyrosine 663 in platelet endothelial cell adhesion molecule-1 trafficking and transendothelial migration. J Immunol. 182, 5041-51 (2009)
  3. Sachs, U.J., Andrei-Selmer, C.L., Maniar, A. et al. The neutrophil-specific antigen CD177 is a counter-receptor for platelet endothelial cell adhesion molecule-1 (CD31). J Biol Chem. 282, 23603-12 (2007). 
  4. Brown, S., Heinisch, I., Ross, E., Shaw, K., Buckley, C.D., Savill, J. Apoptosis disables CD31-mediated cell detachment from phagocytes promoting binding and engulfment. Nature. 418, 200-3 (2002) 
  5. Yeh, J.C., Otte, L.A., Frangos, J.A. Regulation of G protein-coupled receptor activities by the platelet-endothelial cell adhesion molecule, PECAM-1. Biochemistry. 26, 9029-39 (2008)
  6. Bergom, C., Paddock, C., Gao, C., Holyst, T., Newman, D.K., Newman P.J. An alternatively spliced isoform of PECAM-1 is expressed at high levels in human and murine tissues, and suggests a novel role for the C-terminus of PECAM-1 in cytoprotective signaling. J Cell Sci. 121, 1235-42 (2008)
  7. Paddock, C., Lytle, B.L., Peterson, F.C., Holyst, T., Newman, P.J., Volkman, B.F., Newman, D.K. Residues within a lipid-associated segment of the PECAM-1 cytoplasmic domain are susceptible to inducible, sequential phosphorylation. Blood. 117, 6012-23 (2011)
  8. Wang, Y., Su, X., Sorenson, C.M., SheibaniN. Tissue-specific distributions of alternatively spliced human PECAM-1 isoforms. Am J Physiol Heart Circ Physiol. 284, H1008-17 (2003) 
  9. Paddock, C., Zhou, D., Lertkiatmongkol, P., Newman, P.J., Zhu, J. Structural basis for PECAM-1 homophilic binding. Blood. 127, 1052-61 (2016)
  10. Wollscheid, B., Bausch-Fluck, D., Henderson, C., O'Brien, R., Bibel, M., Schiess, R., Aebersold, R., Watts, J.D. Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins. Nat Biotechnol. 27, 378-86 (2009)
  11. Chen, R., Jiang, X., Sun, D., Han, G., Wang, F., Ye, M., Wang, L., Zou, H. Glycoproteomics analysis of human liver tissue by combination of multiple enzyme digestion and hydrazide chemistry. J Proteome Res. 8, 651-61 (2009) 
  12. Famiglietti, J., Sun, J., DeLisser, H.M., Albelda, S.M. Tyrosine residue in exon 14 of the cytoplasmic domain of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) regulates ligand binding specificity. J Cell Biol. 138, 1425-35 (1997)
  13. Hattori, H. Caution should be taken in using CD31 for distinguishing the vasculature of lymph nodes. J Clin Pathol. 56, 638-9 (2003)
  14. Alexander-Sefre, F., Singh, N., Ayhan, A., Salveson, H.B., Wilbanks, G., Jacobs, I.J. Detection of tumour lymphovascular space invasion using dual cytokeratin and CD31 immunohistochemistry. J Clin Pathol. 56, 786-8 (2003) 
  15. Kurtz, K.A., Hoffman, H.T., Zimmerman, M.B., Robinson, R.A. Perineural and vascular invasion in oral cavity squamous carcinoma: increased incidence on re-review of slides and by using immunohistochemical enhancement. Arch Pathol Lab Med. 129, 354-9. (2005)
  16. El-Gohary, Y.M., Metwally, G., Saad, R.S., Robinson, M.J., Mesko, T., Poppiti, R.J. Prognostic significance of intratumoral and peritumoral lymphatic density and blood vessel density in invasive breast carcinomas. Am J Clin Pathol. 129, 578-86. (2008)