Cell surface proteins of the neutrophil in relation to chronic myeloid leukaemia

Waters, J.J. (Jonathan James) (1982) Cell surface proteins of the neutrophil in relation to chronic myeloid leukaemia. PhD thesis, University of Glasgow.

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A large number of studies now suggest that non-random chromosomal changes are associated with human cancers. Certain chromosomes are involved more frequently in rearrangements, duplications and deletions than would be expected by chance. The most consistently observed chromosome abnormality is the Philadelphia chromosome, (Ph') which is found in a number of heamopoetic cell lineages in some 90% of patients sufferring from chronic myeloid leukaemia, (CML). Despite the diagnostic significance of the Ph' chromosome virtually nothing is known about its role in the origin and development of the disease.

Well established phenotypic alterations in the mature neutrophil in CML are limited to the finding of a reduced level of alkaline phophatase in these cells. However, membrane-related phenomena, including phagocytosis and lectin agglutination have been shown to be altered.

This study was undertaken to determine whether simple consistent alterations in cell membrane proteins were detectable at the molecular level which might underlie the membrane-related phenomena noted above and could be related to the chromosomal changes which occur in this leukaemia.

Labelling of the membrane proteins of the cell chosen for study, the neutrophil, revealed at least thirteen relatively high molecular weight polypeptides bands which were identified using the 125I-lactoperoxidase labelling technique, on SDS-PAGE.

To demonstrate that the labelled polypeptides were present on the cell surface, three independent methods were used: plasma membrane isolation, trypsin sensitivity and labelling in the presence and absence of exogenous enzymes.

Extraction with the non-ionic detergent, Triton X-100 considerably reduced problems of handling and proteolytic digestion associated with this cell. Evidence is presented that non-ionic detergents, (TX-100 and NP-40), extract polypeptides selectively which may have structural significance. One major coomassie blue staining band, of mol. wt. 85K, a minor band of mol. wt. 25K and one broad radiolabelled band of mol. wt. 55-60K were particularly selectively retained in a residual pellet.

Experiments using Concanavalin A (Con A) affinity chromatography showed that most of the labelled polypeptides were glycosylated. One major band was unique in that it showed no binding to Con A either by affinity chromatography or by the use of 125I-Con A overlay on SDS-PAGE. It is the major cell surface receptor for Wheat Germ Agglutinin (WGA) which binds to terminal sialic acid residues. when labelled cells were treated with neuraminidase there was an apparent decrease in the mobility of this polypeptide and binding to WGA was abolished. No other labelled band showed significant alteration following neuraminidase treatment.

The above evidence suggests that this glycoprotein of mol. wt. 115K approx., (Gp 115K), is the major sialoglycoprotein at the cell surface of the human neutrophil. This finding may be of general interest since it shares a number of the features, described above, with other 'glycophorin-like' sialoglycoproteins reported in the literature. The function of this unusual class of cell surface glycoproteins is not clear, but they appear to make a significant contribution to the net negative charge at the cell surface and to lack secondary or tertiary structure in the extracellular portion.

Membrane related phenomena of the human neutrophil appear to undergo alterations in CML. Cell surface polypeptides were labelled directly using the 125I-LPO method and radio-labelled lectins (Con A and WGA) were overlayed on whole cell detergent extracts run on SDS-PAGE, in order to try and detect alterations in glycosylation. The results suggest that large scale alterations in the expression of glycosylated, and in particular cel1-surface, proteins do not occur. No evidence for novel gene products or marked alteration in glycosylation of neutrophils in CML was found.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > QH Natural history > QH426 Genetics
Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Ferguson-Smith, Professor Malcolm
Date of Award: 1982
Depositing User: Enlighten Team
Unique ID: glathesis:1982-83202
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 Oct 2022 07:23
Last Modified: 17 Oct 2022 07:23
Thesis DOI: 10.5525/gla.thesis.83202
URI: http://theses.gla.ac.uk/id/eprint/83202

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