Intracellular retention of a factor VIII protein with an Arg2307-->Gln mutation as a cause of haemophilia A.

Substitution of Arg2307 by Gln in factor VIII has been found to be associated with mild to moderate haemophilia A [Gitschier, Wood, Shuman and Lawn (1986) Science 232, 1415-1416]. We have introduced this particular point mutation into a B-domain-deleted factor VIII cDNA and expressed the modified cDNA in C127 cells. Cells expressing the resulting protein, termed des-(868-1562)-factor VIII-R2307Q, were compared with those expressing the previously characterized des-(868-1562)-factor VIII. No immunoreactive material could be detected in the conditioned medium of cells transfected with des-(868-1562)-factor VIII-R2307Q cDNA using assays specific for the factor VIII light chain and the factor VIII heavy chain. Analysis of metabolically labelled cells transfected with des-(868-1562)-factor VIII-R2307Q cDNA revealed that this mutant protein is synthesized at a level similar to des-(868-1562)-factor VIII. In contrast to des-(868-1562)-factor VIII, metabolically labelled des-(868-1562)-factor VIII-R2307Q was not encountered in the conditioned medium of the transfected cells, indicating that the mutant protein is not secreted from the cell. Inspection of the intracellular localization of the two proteins in the cell employing morphological analysis, endoglycosidase H and experiments with inhibitors of glucosidases I and II was consistent with localization of des-(868-1562)-factor VIII and des-(868-1562)-factor VIII-R2307Q in the endoplasmic reticulum. Taken together, our data indicate that the Arg2307-->Gln mutation results in aberrant intracellular trafficking of factor VIII, which may explain the low levels of factor VIII antigen in the plasma of haemophilia A patients that carry this particular point mutation.