Secretion of Human Furin into Mouse Milk

Abstract

We have previously described the expression of the human proprotein convertase furin or paired basic amino acid-cleaving enzyme, in mice transgenic for paired basic amino acid-cleaving enzyme and human Protein C (HPC). Here we show 100-fold or higher expression of furin in the mammary gland, compared with endogenous furin. Furin and recombinant HPC were detected in the same regions of the mammary gland and regulated similar to the endogenous whey acidic protein. In addition to the expected intracellular localization, furin was secreted into the milk as an 80-kDa form lacking the transmembrane and cytoplasmic domains. Furin present at levels of up to 40,000 units/ml milk cleaved the t-butoxycarbonyl-RVRR-AMC substrate with a K m of 32 M, and processed the recombinant HPC precursor at the appropriate sites. Surprisingly, the expression of an active protease was not toxic to the mammary gland. This is a rare example of an animal model secreting active truncated forms of a processing endoprotease into a bodily fluid.Furin or paired basic amino acid cleaving enzyme (PACE) 1 is a Ca 2ϩ -dependent serine protease that processes proteins in the constitutive secretory pathway (1). Furin belongs to a family of subtilisin-like proprotein convertases including kexin (EC 3.4.21.61), PC1/3, PC2, PC4, PC5/6, PACE4 (reviewed in Ref. 1), and PC7/PC8 or lymphoma proprotein convertase (2-5), that play a role in the maturation of proproteins by cleavage at dibasic or tetrabasic sites (6). Unlike PC1/3, PC2, and PC4, which are restricted to endocrine, neural, or testicular germ cells and ovaries, respectively, the furin gene is expressed in most cells and tissues examined (1, 7).cDNAs cloned from several species show furin to contain prepro-, catalytic and middle domains, a cysteine-rich region, transmembrane anchor, and cytoplasmic domain (1). Furin is activated by autocatalytic cleavage of its propeptide, presumably at the -Lys-Arg-Arg-Thr-Lys-Arg Ϫ1 -2 site (1,8). A membrane-associated protein, it is found mainly in the trans-Golgi network (TGN) co-localized with the TGN 38 marker protein (9). Some amount of furin recycles between the cell surface and the TGN (9) in endosome-like structures (10). So far furin has not been detected in bodily fluids like blood, nor has the effect of its expression on cell and organ development been studied in transgenic animals.Transgenic animals secreting recombinant proteins into milk, blood, urine, or saliva (11) have been generated and differences in recombinant protein modification compared with the human counterpart observed (12). In a pioneering attempt to engineer the post-translational capacity of the mouse mammary gland, we reported enhanced proteolytic maturation of the recombinant human Protein C (rHPC) precursor upon coexpression with furin (13). However, the effect of increased furin concentration on mammary gland development could not be predicted, nor its' intra-and extracellular distribution. Here we demonstrate localization of two heterologous proteins with the endogenous whey ac...

Comparative Characterization of Two Forms of Recombinant Human SPC1 Secreted from Schneider 2 Cells

“…Analysis of kinetic data by Hill plot revealed no cooperativity for both forms (0.99 for hSPC1/714 and 1.00 for hSPC1/585), implying that hSPC1 essentially behaves as a monomer. The kinetic parameters presented here are similar to those obtained using other expression systems (15)(16)(17) suggesting that hSPC1 produced by S2 cells is enzymatically equivalent to hSPC1 produced in mammalian cells.…”

“…In order to generate enzymatically active SPC1, numerous expression systems have been used in the past; these include recombinant vaccinia virus (15), recombinant baculovirus-infected insect cells (16), and secretion in transgenic murine milk (17). These systems are not always easy to manipulate and lead to varying levels of expressed protein and, finally, purification of proteins of interest in their native state is sometimes compromised because of cell lysis.…”

The microenvironment patterns the pluripotent mouse epiblast through paracrine Furin and Pace4 proteolytic activities

“…The acidic pH of the trans-Golgi network/endosomal system favors a second autocatalytic cleavage displacing an inhibitory propeptide (Anderson et al 2002), and cleavage of the membrane anchor by an unknown ''sheddase'' in acidic compartments can give rise to a soluble form of activated Furin (Wise et al 1990;Vidricaire et al 1993;Denault et al 2002). Shed Furin has been observed in the milk of transgenic mice (Paleyanda et al 1997) and in epididymal fluid (Thimon et al 2006). Soluble Furin (or Pace4) can also activate Nodal in a complex with its coreceptor, Cripto, in cultured cells (Blanchet et al 2008).…”

Furin as proprotein convertase and its role in normal and pathological biological processes

“…Однако при удалении трансмембранного и цитоплазматического доменов с С-конца молекулы в модельных системах укороченная рекомбинантная форма становится растворимой. Эта форма обладает полной протеолитической активностью и широко используется для исследования энзиматических, кинетических и других свойств фермента [8]. В 2003 году растворимая форма фурина мыши была закристаллизована в комплексе с необратимым ингибитором.…”