Background

Recently a missense mutation c.787A>T truncating CD177 protein has been introduced to regulate the absence of CD177 protein on the neutrophil surface of CD177-null individuals. Despite this, not all CD177-null individuals are homozygous for c.787T.

Here we investigated if c.1291G>A polymorphism by de-stabilizing CD177 GPI-anchor sequence, converts membrane-bound CD177 to a soluble form and leads to the absence of CD177 from the neutrophil surface in heterozygote c.787T CD177-null individuals.

Methods

To prove this hypothesis, stably transfected cells HEK293F cells expressing CD177 wildtype (c.1291G) and mutant c.1291A were produced.

The expression of CD177 in both forms in transfected cells was analyzed by immunoprecipitation and immunoblot. The membrane expression of CD177 in both allelic forms was evaluated in flow cytometry. The reactivities of CD177 alloantibodies with immobilized CD177 (in both forms) were assessed in ELISA.

In confocal laser scanning microscopy, co-transfected COS-7 cells expressing fluorescence labeled CD177 (wildtype and c.1291A mutant) and PR3, the co-localization of both CD177 allelic forms with PR3 was evaluated.

Results

Cell lysate analysis detected CD177 protein in cytoplasm of both transfected cells. However, flow cytometry analysis detected CD177 protein only on the surface of CD177 wildtype expressing cells but not c.1291A mutant.

Using anti-V5 mab, CD177 in both forms was precipitated from the cell culture medium. In ELISA comparative reactivity of anti-HNA-2 alloantibodies with both immobilized CD177 forms was detected.

In confocal laser microscopy analysis of COS-7 cells co-transfected with fluorescence-labeled CD177 (wildtype and c.1291A mutant) and PR3, CD177 (in both allelic forms) were detected with PR3 in the cytoplasm. However, on the cell surface PR3 was detectable only when cells were co-transfected with CD177 wildtype.

Conclusion

Here we describe a new allele of CD177 protein encoded by CD177 c.1291A. In comparison to CD177 wildtype, c.1291A allelic form showed decreased membrane stability and increased solubility. However, in ELISA, a comparable reactivity of CD177 alloantibodies with both proteins was detected indicating a similar conformational structure for both proteins. This data explain the absence of CD177 on the neutrophil surface of CD177-null individuals heterozygous for c.787T allele who carry c.1291A allele.

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