We identified a novel Drosophila protein of ≈ 400kDa, hemolectin (d-Hml), secreted from haemocyte-derived Kc167 cells. Its 11.7kbp cDNA contains an open reading frame of 3843 amino acid residues, with conserved domains in von Willebrand factor (VWF), coagulation factor V/VIII and complement factors. The d-hml gene is located on the third chromosome (position 70C1-5) and consists of 26 exons. The major part of d-Hml consists of well-known motifs with the organization: CP1-EG1-CP2-EG2-CP3-VD1-VD2-VD′-VD3-VC1-VD′′-VD′′′-FC1-FC2-VC2-LA1-VD4-VD5-VC3-VB1-VB2-VC4-VC5-CK1 (CP, complement-control protein domain; EG, epidermal-growth-factor-like domain; VB, VC, VD, VWF type B-, C- and D-like domains; VD′, VD′′, VD′′′, truncated C-terminal VDs; FC, coagulation factor V/VIII type C domain; LA, low-density-lipoprotein-receptor class A domain; CK, cysteine knot domain). The organization of VD1-VD2-VD′-VD3, essential for VWF to be processed by furin, to bind to coagulation factor VIII and to form interchain disulphide linkages, is conserved. The 400kDa form of d-Hml was sensitive to acidic cleavage near the boundary between VD2 and VD′, where the cleavage site of pro-VWF is located. Agarose-gel electrophoresis of metabolically radiolabelled d-Hml suggested that it is secreted from Kc167 cells mainly as dimers. Resembling VWF, 7.9% (305 residues) of cysteine residues on the d-Hml sequence had well-conserved positions in each motif. Coinciding with the development of phagocytic haemocytes, d-hml transcript was detected in late embryos and larvae. Its low-level expression in adult flies was induced by injury at any position on the body.
Abbreviations used: b-Hmc, Bombyx mori haemocytin; d-Hml, Drosophila hemolectin; RT-PCR, reverse transcriptase PCR; TCA, trichloroacetic acid; VWF, von Willebrand factor; CK, cysteine knot domain; CP, complement-control protein domain; VD, VWF type D-like domain; VD′, VD′′ and VD′′′, truncated C-terminal VDs; VC, VWF type C-like domain; EG, epidermal-growth-factor-like domain; LA, low-density-lipoprotein receptor class A domain.
Present address: Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan.
Present address: Institut de Biologie et Chimie des Proteins, UPR 412 CNRS, 7, Passage du Vercors-69367 Lyon Cedex 07, France.
The nucleotide sequence data reported here are deposited in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number AB035891 and in the Berkeley Drosophila Genome Projects database with Gadfly number CG7002.