cells secrete CfaD, a protein that is similar to cathepsin proteases. starving cells, as indicated by a high concentration of CMF (Jain et al., 1992; Yuen et al., 1995), the cells aggregate using relayed pulses of extracellular cAMP as a chemoattractant (Aubry and Firtel, 1999). The aggregating cells form streams that break up into groups ICG-001 of ~20,000 cells (Shaffer, 1957). Each group develops into a fruiting body consisting of a mass of spore cells supported on a ~1-mm-high column of stalk cells. A secreted ~450 kDa protein complex that is called counting factor (CF) modulates adhesion and motility during aggregation to regulate stream break-up, and thus group- and fruiting-body size (Brock and Gomer, 1999; Gao et al., 2004; Jang and Gomer, 2005; Roisin-Bouffay et al., 2000; Tang et al., 2002). We found that AprA, a 60 kDa protein in a partially purified CF preparation, is not a CF component but, rather, is part of a ~150 kDa complex that inhibits proliferation and, thus, has the properties of a chalone (Brock and Gomer, 2005). Here, we show that another protein, CfaD, is also not a component of CF. Instead, CfaD is part of a ~150 kDa complex, interacts with AprA and, similar to AprA, has the properties of a chalone. Results CfaD is a cathepsin-L like protein but lacks the protease activity Some preparations of partially purified CF contained a 27 kDa protein. The amino acid (aa) sequence of a tryptic peptide of this protein matched part of an open reading frame in the genome (supplementary material Fig. S1). We named the predicted protein CfaD for CF-associated protein. The predicted molecular mass of CfaD is 58.6 kDa, suggesting that the 27 kDa protein is a breakdown fragment of CfaD. The predicted CfaD aa sequence MMP2 contains a peptidase C1A motif and is, over a stretch of 315 aas, 34% similar to cathepsin L precursors from the mosquito and other species (supplementary material Fig. S2). Cathepsins are a family of proteases responsible for protein turnover in the lysosome (Nomura and Katunuma, 2005). Tumors often contain increased levels of cathepsins and, unlike normal cells, secrete cathepsins, which appear to promote invasion by degrading the surrounding extracellular matrix (Gocheva and Joyce, 2007; Jedeszko and Sloane, 2004). CfaD also shows 34% similarity to the 26/29 kDa proteinase of the flesh fly (supplementary material Fig. S2), which is synthesized as a ~62 kDa polypeptide with a 19-aa signal sequence. This protein can hydrolyse the cathepsin substrate Z-Phe-Arg-AMC (Fujimoto et al., 1999). During the processing of the 26/29 kDa proteinase, the signal sequence is removed and the remaining protein ICG-001 is cleaved into a 23 kDa and an ~25 kDa fragment, whereby the 13 kDa fragment of the precursor that lies between the 23 kDa and 25 kDa fragments is then discarded (Fujimoto et al., 1999). Both the 23 and 25 kDa subunits are post-translationally glycosylated, and the resulting 26 kDa and 29 kDa fragments are secreted by hemocytes into the hemolymph of larvae to degrade the larval midgut and fat body during metamorphosis (Fujimoto et al., 1999; Nakajima et al., 1997; Takahashi et al., 1993). In CfaD, there is a predicted 18-aa signal sequence, and the aa sequence of a tryptic peptide of the secreted form of CfaD begins at ICG-001 the predicted signal sequence cleavage site (supplementary material Fig. S1, arrow), suggesting that the secreted form of the 27 kDa fragment of CfaD (CfaD-27) begins with VPQL. A comparison of the predicted CfaD aa sequence with other cathepsin sequences (Berti and Storer, 1995; Santamaria et al., 1998) indicated that CfaD contains two key active site residues, a glutamine at position 327 and a cysteine at position 333 (supplementary material Figs S1, S2). However, CfaD belongs to the peptidase C1 family, which includes proteins without peptidase activity (Rawlings and Barrett, 1993). Using the protease assay that showed that the 26/29-kDa proteinase has a protease activity (Fujimoto et al., 1999), we observed that in PBM (roughly mimicking the extracellular environment), a human cathepsin-L control had activities of ~2.5 and ~3.1 nM Z-Phe-Arg-AMC hydrolyzed/hour/g protein at 22C and 37C, respectively. We observed that rCfaD (recombinant CfaD containing a His tag), rHMCfaD (recombinant CfaD containing both His and Myc tags), and rHMCfaD-PM (rHMCfaD with Gln327 changed to Lys and Cys333 changed to Gly) had no detectable protease activity at either 22C or 37C, with ICG-001 a detection limit ICG-001 of 0.004 nM Z-Phe-Arg-AMC hydrolyzed/hour/g protein. In addition, rCfaD had no detectable protease activity at pH 5.2, roughly corresponding to the pH within.