Tissue expression of copines and isolation of copines I and III from the cytosol of human neutrophils

Jack B. Cowland^*, Daniel Carter^*, Malene D. Bjerregaard^*, Anders H. Johnsen, Niels Borregaard^* and Karsten Lollike^*^,1

The Granulocyte Research Laboratory, Departments of
^* Hematology and
Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark

¹Correspondence at current address: Novo Nordisk Health Care AG, Andreasstrasse 15, 8050 Zurich, Switzerland. E-mail: kalo{at}novonordisk.com

Copines are a recently identified group of proteins characterizedby two Ca²⁺-binding C2-domains at the N terminus and an A-domainat the C terminus. Although pEST sequences indicate the existenceof at least seven copines in man, only copines I, III, and VIhave been identified at protein level. Here, we describe theisolation of copines I and III in the cytosol of human neutrophilsby use of Ca²⁺-induced hydrophobic chromatography. This is thefirst demonstration that copines are coexpressed in the samecell. We found that copine III exists in the cytosol of humanneutrophils as a monomer with a blocked N terminus. CopinesI and III undergo conformational changes upon Ca²⁺ binding thatlead to exposure of hydrophobic patches. Examination of RNAfrom 68 human tissues demonstrated that copines I–IIIare ubiquitously expressed whereas copines IV–VII eachhas a more restricted and individual expression profile. Expressionof copines I–III was also demonstrated in neutrophil precursorsfrom bone marrow. Copine I was uniformly expressed at all stagesof neutrophil differentiation, whereas copine II and even moreso, copine III were expressed in the more immature neutrophilprecursors, which indicates an individual function of thesecopines.

Key Words: granulopoiesis • calcium-binding proteins • mRNA profiles • differentiation