Identification and cloning of the SNARE proteins VAMP-2 and syntaxin-4 from HL-60 cells and human neutrophils

• Published in: Inflammation 2001-08, Vol.25 (4), p.255-265
• Main Authors: Smolen, J E, Hessler, R J, Nauseef, W M, Goedken, M, Joe, Y
• Format: Article
• Language: English
• Subjects: Cell Differentiation - genetics; DNA, Complementary - genetics; DNA, Complementary - isolation & purification; Granulocytes - cytology; Granulocytes - metabolism; HL-60 Cells; Humans; Membrane Proteins - analysis; Membrane Proteins - genetics; Nerve Tissue Proteins - analysis; Nerve Tissue Proteins - genetics; Neutrophils - cytology; Neutrophils - metabolism; Qa-SNARE Proteins; R-SNARE Proteins; RNA, Messenger - analysis; SNARE protein; SNARE Proteins; syntaxin-4; VAMP-2 protein; Vesicular Transport Proteins
• ISSN: 0360-3997; 1573-2576
• DOI: 10.1023/A:1010903804063

Degranulation and membrane fusion by neutrophils are essential to host defense. We sought homologues of neuron-specific fusion proteins in human neutrophils and in their precursors, the promyelocytic cell line HL-60. We screened a differentiated HL-60 library and obtained an 848 bp sequence with a 351 bp open reading frame, identical to that published for human VAMP-2 and including 5' and 3' untranslated regions. RNA from HL-60 cells during differentiation into the neutrophil lineage was subjected to Northern blot analysis. which revealed a transcript of approximately 1050 bp at all stages of differentiation. The amount of these transcripts increased approximately threefold during differentiation, a finding confirmed by quantitative RT-PCR. We also detected mRNA for VAMP-2 in human neutrophils and monocytes using RT-PCR. In like fashion, transcripts of syntaxin-4, another fusion protein, were recovered from a neutrophil cDNA library. As with VAMP-2, expression of syntaxin-4 (determined by Northern blots) also increased, but by only 50%, during differentiation of HL-60 cells. These studies demonstrate that neutrophils and their progenitors possess mRNA for the fusion proteins VAMP-2 and syntaxin-4, and that their transcription increases during differentiation, concurrent with the functional maturation of myeloid cells.