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Purification and characterization of a 95 kDa protein: from normal human granulocytes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
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2002 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 270, no 1, 27-35 p.Article in journal (Refereed) Published
Abstract [en]

A 95-kDa protein was purified to homogeneity from granule extracts of normal human granulocytes. The column procedure consisted of Sephadex G-75, Mono-S cation exchange and Superdex HR 75 chromatography. The purified protein showed only one broad band at a molecular weight of 95 kDa when analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It reacted with polyclonal antibodies against carcinoembryonic antigen (CEA) and a specific monoclonal antibody against CD66b, but did not react with monoclonal antibodies against CD66acde and CD66c when analyzed by immunoblotting. The molecular weight of the protein shifted from 95 to 40 kDa on SDS-PAGE after deglycosylation. Tryptic peptide analysis by MALDI-Tof identified four peptides with spectra of m/z matching the expected tryptic peptides from a CGM6 gene product. Furthermore, the nanoelectrospray mass spectrometry (MS/MS) analysis of the two selected tryptic peptides of the protein revealed two amino acid sequences corresponding to residues 79-98 and 199-207 of the CGM6 gene product. Based on this, and also on the immunochemical data, it is concluded that the purified 95 kDa is identical to carcinoembryonic antigen-related cell adhesion molecule 8 (CEACAM8) (nonspecific cross-reacting antigen (NCA)-95, CD67 and CD66b) and is a product of the CGM6 (W272) gene. We present, for the first time, a method for the purification of CEACAM8 from normal human granulocytes, which should be useful for further studies on its structure and functions. We also confirmed at the protein level that CEACAM8 is a product of the CGM6 (NCA-W272) gene.

Place, publisher, year, edition, pages
2002. Vol. 270, no 1, 27-35 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-92100DOI: 10.1016/S0022-1759(02)00215-6PubMedID: 12379336OAI: oai:DiVA.org:uu-92100DiVA: diva2:165058
Available from: 2004-09-20 Created: 2004-09-20 Last updated: 2010-11-22Bibliographically approved
In thesis
1. CarcinoEmbryonic Antigen-related Cell Adhesion Molecule 8 (CEACAM8): Purification, Characterization, Cellular and Clinical Studies
Open this publication in new window or tab >>CarcinoEmbryonic Antigen-related Cell Adhesion Molecule 8 (CEACAM8): Purification, Characterization, Cellular and Clinical Studies
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A 95-kDa protein was purified from normal human granulocytes. The protein reacted with a monoclonal antibody against CEACAM8. MALDI-Tof and MS/MS analyses revealed the protein to be a CGM6 gene product. Thus, the protein was proved to be identical to CEACAM8.

An ELISA for CEACAM8 was developed with detection range of 1-64μg/L. Data are presented on the levels of CEACAM8 in the blood of healthy individuals and patients undergoing surgery, as well as in patients with acute infection. The highly elevated levels of CEACAM8 in the blood of these patients were significantly correlated with the surface expression of CEACAM8 on neutrophils and the number of circulating neutrophils, which suggests that CEACAM8 could serve as a biological marker for granulocyte activitiy in vivo.

The cellular content of CEACAM8 in neutrophils was estimated to be 82.4 ± 8.9 ng/106 cells. Subcellular localisation and mobilisation studies showed that the majority of CEACAM8 is present in the secondary granules of human neutrophils, with a small amount on the plasma membranes. Upon stimulation, CEACAM8 translocated to the plasma membranes from the secondary granules and was also released extracellularly (5.5 ± 0.7% of the total content of CEACAM8).

In eosinophils, the cellular content of CEACAM8 was estimated to be 73.8 ± 6.0 ng/106 cells. In these cells, CEACAM8 is mainly stored in secretory vesicles. Upon activation, eosinophils released 5.1 ± 1.1% of the total content of CEACAM8.

Administration of granulocyte colony-stimulating factor (G-CSF) to healthy individuals resulted in an increased content of CEACAM8 in neutrophils on day 1, and decreased on day 4. However, the content of CEACAM8 in light membrane fractions was increased on day 4. The translocation of CEACAM8 observed in vivo after G-CSF administration is probably not directly related to this cytokine but to other cytokines such as TNF-a.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 64 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1372
Biochemistry, human neutrophil, human eosinophil, granule proteins, CEA, CEACAM8, G-CSF, ELISA, subcellular fractionation, Biokemi
National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-4534 (URN)91-554-6031-3 (ISBN)
Public defence
2004-10-15, Rosensalen, entrance 95/96, Akademiska sjukhuset, Uppsala, 13:15
Available from: 2004-09-20 Created: 2004-09-20Bibliographically approved

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