RESUMO
Leukophysin (LKP) is a 28-kDa protein of CTL and U937 monocytic cells that is located in the membrane of high density granules as well as lighter cytoplasmic granules or vesicles. mAbs to KLP were used to clone a full length cDNA clone with an open reading frame coding for a 235-amino acid polypeptide with a molecular mass of 24.3 kDa and two potential transmembrane regions. The nucleotide sequence was highly homologous to the 3' end of human RNA helicase A. Expression of the LKP was confirmed as a reverse transcriptase-PCR product that may be an alternately spliced product of RNA helicase A. The cDNA contained a repetitive motif that was similar to synaptophysin 1, a protein that is important for synaptic vesicle exocytosis. A polyclonal Ab directed against the 17 carboxyl-terminal amino acids of LKP detected the same 28-kDa granule membrane protein as the D545, one of the mAbs used to clone the cDNA. In addition, the D545 mAb reacted strongly with the GST fusion protein of the bacterially expressed LKP cDNA. In confocal immunofluorescence studies, the anti-LKP peptide Ab reacted with granzyme A-negative granules and vesicles in CD8+ CTL lymphocytes from normal and Chediak-Higashi patients. Thus, based on the expression of the C-terminal LKP epitope, vesicular structures an granules have been detected in CTL that are distinct from classical granzyme-containing cytolytic granules.
Assuntos
Grânulos Citoplasmáticos/enzimologia , Membranas Intracelulares/enzimologia , Proteínas de Membrana/isolamento & purificação , Proteínas de Neoplasias/isolamento & purificação , RNA Nucleotidiltransferases/genética , Linfócitos T Citotóxicos/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Clonagem Molecular , RNA Helicases DEAD-box , DNA Complementar/isolamento & purificação , Epitopos/análise , Granzimas , Humanos , Leucemia de Células T , Linfoma Difuso de Grandes Células B , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , RNA Helicases , RNA Nucleotidiltransferases/análise , Homologia de Sequência do Ácido Nucleico , Serina Endopeptidases/análise , Células Tumorais CultivadasRESUMO
A membrane glycoprotein of human platelet dense granules, called granulophysin, with serologic homology to synaptophysin has recently been identified. To determine if this protein was present in granulated leukocytes, we examined several cell types for the presence of the protein by indirect immunofluorescence. Antigranulophysin mAb staining was detected in a granular pattern in the cytoplasm of permeabilized IL-2-stimulated CD3+ peripheral lymphocytes, neutrophils, U937 monocytes, and mast cells. Immunohistochemistry of human lymph nodes showed cytoplasmic staining of macrophages, neutrophils, and some dendritic cells. Induction of granule exocytosis in granulated CD3+ lymphocytes after stimulation with PMA and calcium ionophore A23187 resulted in a redistribution of the reactive epitope from the cytoplasm to the plasma membrane. Subcellular fractions contained two peaks of reactivity; the first peak coincided with N-benzyloxycarbonyl-L-lysine thiobenzyl ester-esterase activity in dense granules whereas the second peak was present in lighter fractions. The affinity purified protein from both peaks was identical in Western blot analysis and had a molecular mass of 28 kDa under reducing conditions. The protein could only be solubilized in detergent suggesting that it was an integral membrane protein. We have named this protein leukophysin to differentiate it from the 40-kDa granulophysin of platelets. Monocytes contained a protein with identical m.w. to leukophysin, whereas a protein of a slightly higher m.w. was detected in neutrophils. We propose that leukophysin is a common granule membrane protein of leukocytes.
