A. J. M. Roebroek

Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes

The human fur gene encodes a protein, designated furin, the C-terminal half of which contains a transmembrane and a cysteine-rich receptor-like domain. The N-terminal half of furin exhibits striking primary amino acid sequence similarity to the catalytic domains of members of the subtilisin family of serine proteases. We here report characteristics of the furin protein and propose a three-dimensional model for its presumptive catalytic domain with characteristics, that predict furin to exhibit an endo-proteolytic cleavage selectivity at paired basic residues. This prediction is substantiated by transfection and cotransfection experiments, using COS-1 cells. Full length fur cDNA evokes the specific synthesis of two polypeptides of about 100 kDa and 90 kDa as appeared from Western blot analysis of transfected COS-1 cells using a polyclonal anti-furin antiserum. Functional analysis of furin was performed by cotransfection of fur cDNA with cDNA encoding the ‘wild type’ precursor of von Willebrand factor (pro-vWF) and revealed an increased proteolytic processing of prov WF. In contrast, cotransfection of fur cDNA with a recombinat derivative (provWFgly763), having the arginine residue adjacent to the proteolytic cleavage site (arg-ser-lys-arg) replaced by glycine, revealed that provWFgly763 is not processed by the fur gene product. We conclude that in higher eukaryotes, furin is the prototype of a subtilisin-like class of proprotein processing enzymes with substrate specificity for paired basic residues.

Evolutionary conserved close linkage of the c-fes/fps proto-oncogene and genetic sequences encoding a receptor-like protein.

Recently we described that genetic sequences in the immediately upstream region of the c‐fes/fps proto‐oncogene, designated fur, constituted a transcription unit for a 4.5‐kb mRNA. Here we present characteristics of the genetic organization of fur and some features of its putative translation product which we call furin. The nucleotide sequence of a 3.1‐kbp fur‐specific cDNA isolated from a human cDNA library revealed an open reading frame of 1,498 bp from which the 499 carboxy‐terminal amino acids of the primary fur translational product could be deduced. Computer analysis indicated that furin contained a possible transmembrane domain which resembled that of class II MHC antigens. Furthermore, a cysteine‐rich region was present. Significant homology, especially with respect to the topography of cysteine residues, was found between the cysteine‐rich regions of the human insulin receptor, the human epidermal growth factor receptor and furin. From the data presented here we deduce that fur may encode a membrane‐associated protein with a recognition function.

fur gene expression as a discriminating marker for small cell and nonsmall cell lung carcinomas.

The recently discovered fur gene encodes a membrane-associated protein with a recognition function. To further characterize the gene, we studied its expression by Northern blot analysis using poly(A)-selected RNA from a variety of organs of African green monkey and rat. The fur gene appeared to be differentially expressed, relatively high levels of fur mRNA being present in specimens of liver and kidney, low levels in brain, spleen, and thymus, and very low levels in heart muscle, lung, and testis. mRNA levels in specimens of human lung tissue without neoplastic lesions were also very low. Similar analyses of primary human lung carcinomas of different histopathological types revealed a highly selective and strong elevation of fur expression in nonsmall cell lung carcinomas, but not in small cell lung carcinomas. These results indicate that fur expression can be used to discriminate between these two types of human lung cancer.

The structure of the human c-fes/fps proto-oncogene.

We have determined the complete nucleotide sequence of a human DNA fragment of approximately 13 kbp, which was shown by Southern blot analysis to contain the entire v‐fes/fps cellular homolog. The v‐fes/fps homologous sequences were dispersed over 11 kbp in 18 interspersed segments which were flanked by splice junctions. Fusion of these segments created a DNA fragment in which coding regions similar to those observed in the viral oncogenes v‐fes of the Gardner‐Arnstein (GA) and Snyder‐Theilen (ST) strains of feline sarcoma virus and v‐fps found in Fujinami sarcoma virus could be identified. A potential initiation site in the first exon was found. About 200 nucleotides downstream of a translational stop codon in the v‐fes/fps homologous region, a poly(A) addition signal was identified. The deduced amino acid sequence has a molecular weight of 93 390 dalton resembling NCP92, the recently described human c‐fes/fps product. The topography of human c‐fes/fps appeared to resemble that of chicken c‐fps.

Generation of structural and functional diversity in furin-like proteins in Drosophila melanogaster by alternative splicing of the Dfur1 gene.

To investigate whether or not alternative splicing might be a mechanism by which in Drosophila melanogaster diversity is generated in endoproteases of the novel eukaryotic family of subtilisin‐like proprotein processing enzymes, we determined structural and functional characteristics of the Dfur1 gene. Northern blot analysis revealed Dfur1 transcripts of 7.6, 6.5, 4.5 and 4.0 kb. By comparative nucleotide sequence analysis of Dfur1 genomic and cDNA clones, 10 coding exons were identified and, together with Northern blot analysis using exon‐specific probes, evidence was obtained that the four transcripts are generated by alternative splicing and polyadenylation. The apparently complete open reading frames of three Dfur1 cDNAs revealed that these coded for three furin‐like proteins, Dfurin1 (892 residues), Dfurin1‐CRR (1101 residues) and Dfurin1‐X (1269 residues), which possessed common but also unique structural domains. These various isoforms of furin in Drosophila were characterized in gene transfer studies using immunoprecipitation analysis. Differential expression of Dfur1 transcripts was found in Northern blot analysis of RNA from various developmental stages of Drosophila. RNA in situ hybridization experiments revealed that the Dfurin1‐X and Dfurin1‐CRR isoforms are expressed in non‐overlapping sets of tissues during Drosophila embryogenesis. In gene transfer experiments in which the Dfurin1, Dfurin1‐CRR and Dfurin1‐X proteins were expressed at high levels together with the precursor of the beta A‐chain of activin‐A, a member of the transforming growth factor beta (TGF beta) superfamily, or the precursor of von Willebrand factor, all three proteins appeared capable of processing these substrates. Our studies indicate that the Dfur1 gene encodes structurally different subtilisin‐like proprotein processing enzymes with distinct physiological functions in Drosophila.

Furin-mediated proprotein processing activity: Involvement of negatively charged amino acid residues in the substrate binding region

Furin, which is encoded by the recently discovered FUR gene, appears to be the first known mammalian member of the subtilisin family of serine proteases with cleavage selectivity for paired or multiple basic residues. A consensus cleavage sequence, Arg-X-Lys/Arg-Arg has been proposed. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Homology modelling of the catalytic domain of this protein suggested that negatively charged amino acid residues near or in the substrate binding region might contribute to the observed specificity for substrate segments with paired and multiple basic amino acid residues. To investigate this hypothesis, furin mutants were generated in which negatively charged residues, predicted to be located near or in the substrate binding pockets and involved in interactions with basic residues of the substrate, were replaced by neutral residues. Analysis of processing by these furin mutants of wild-type and cleavage mutants of pro-von Willebrand factor (pro-vWF) revealed that particular negatively charged residues are critical for specific cleavage activity.

Regional mapping of the human NSP gene to chromosome region 14q21→q22 by fluorescence in situ hybridization analysis

Genetic sequences of the novel NSP gene, which encodes neuroendocrine-specific proteins, were isolated from cDNA libraries constructed with mRNA isolated from human lung carcinoma cells. Hybridization analysis of a panel of human x mouse cell hybrids with an 0.8-kb NSP cDNA probe indicated that the human NSP gene is probably located on chromosome 14. Fluorescence in situ hybridization analysis of metaphase chromosomes using overlapping genomic clones of NSP as a probe localized the NSP gene to chromosome region 14q21-->q22.

Assignment of the human proprotein convertase gene PCSK5 to chromosome 9q21.3

The human PCSK5 gene, which encodes a subtilisin-like proprotein processing enzyme, has been mapped by analysis of somatic cell hybrids and YAC clones as well as fluorescence in situ hybridization to chromosome 9q21.3 near markers D9S175 and D9S276.