Inhibition of the CaaX proteases Rce1p and Ste24p by peptidyl (acyloxy)methyl ketones

The CaaX proteases Rce1p and Ste24p can independently promote a proteolytic step required for the maturation of certain isoprenylated proteins. Although functionally related, Rce1p and Ste24p are unrelated in primary sequence. They have distinct enzymatic properties, which are reflected in part by their distinct inhibitor profiles. Moreover, Rce1p has an undefined catalytic mechanism, whereas Ste24p is an established zinc-dependent metalloprotease. This study demonstrates that both enzymes are inhibited by peptidyl (acyloxy)methyl ketones (AOMKs), making these compounds the first documented dual specificity inhibitors of the CaaX proteases. Further investigation of AOMK-mediated inhibition reveals that varying the peptidyl moiety can significantly alter the inhibitory properties of AOMKs toward Rce1p and Ste24p and that these enzymes display subtle differences in sensitivity to AOMKs. This observation suggests that this compound class could potentially be engineered to be selective for either of the CaaX proteases. We also demonstrate that the reported sensitivity of Rce1p to TPCK is substrate-dependent, which significantly alters the interpretation of certain reports having used TPCK sensitivity for mechanistic classification of Rce1p. Finally, we show that an AOMK inhibits the isoprenylcysteine carboxyl methyltransferase Ste14p. In sum, our observations raise important considerations regarding the specificity of agents targeting enzymes involved in the maturation of isoprenylated proteins, some of which are being developed as anti-cancer therapeutic agents.     

A Novel Role of the Yeast CaaX Protease Ste24 in Chitin Synthesis

Ste24 is a membrane-integral CaaX metalloprotease residing in the endoplasmic reticulum (ER). In yeast, the only known substrate of Ste24 is the mating factor a precursor. A global screening for protein–protein interactions indicated that Ste24 interacts with chitin synthesis deficient (Chs)3, an enzyme required for chitin synthesis. We confirmed this interaction by yeast two-hybrid analyses and mapped the interacting cytoplasmic domains. Next, we investigated the influence of Ste24 on chitin synthesis. In sterile (ste)24Δ mutants, we observed resistance to calcofluor white (CFW), which was also apparent when the cells expressed a catalytically inactive version of Ste24. In addition, ste24Δ cells showed a decrease in chitin levels and Chs3-green fluorescent protein localized less frequently at the bud neck. Overexpression of STE24 resulted in hypersensitivity to CFW and a slight increase in chitin levels. The CFW phenotype of ste24Δ cells could be rescued by its human and insect orthologues. Although Chs3 binds to Ste24, it seems not to be a substrate for this protease. Instead, our data suggest that Chs3 and Ste24 form a complex in the ER that facilitates protease action on prenylated Chs4, a known activator of Chs3 with a C-terminal CaaX motif, leading to a more efficient localization of Chs3 at the plasma membrane.     

Identification of a human cDNA encoding a novel protein structurally related to the yeast membrane-associated metalloprotease, Ste24p

Recently, a novel membrane-associated metalloprotease, designated Ste24p, has been identified in Saccharomyces cerevisiae [K. Fujimura-Kamada, F.J. Nouvet, S. Michaelis, J. Cell Biol. 27 (1997) 271-285]. We cloned a human brain cDNA encoding a protein homologous to Ste24p (designated Hs Ste24p). The predicted 475-amino acid product of its open reading frame exhibited 62% similarity to Ste24p, and contained a zinc metalloprotease motif (HEXXH) and multiple predicted membrane spans. Northern blot analysis showed that this gene was expressed in most tissues. Immunofluorescence analysis of epitope-tagged Hs Ste24p constructs suggested that it is localized in the ER and possibly also in the Golgi compartment. A search of the expression sequence tag database identified a fragment of DNA encoding a segment homologous to the segment of Hs Ste24p containing the HEXXH motif in insects and nematodes. Thus, Hs Ste24p could be a member of a new family of Ste24p-like membrane-associated metalloproteases which are widely conserved in eukaryotes.     

Reconstitution of the Ste24p-dependent N-terminal proteolytic step in yeast a-factor biogenesis

The yeast mating pheromone a-factor precursor contains an N-terminal extension and a C-terminal CAAX motif within which multiple posttranslational processing events occur. A recently discovered component in a-factor processing is Ste24p/Afc1p, a multispanning endoplasmic reticulum membrane protein that contains an HEXXH metalloprotease motif. Our in vivo genetic characterization of this protein has demonstrated roles for Ste24p in both the N-terminal and C-terminal proteolytic processing of the a-factor precursor. Here, we present evidence that the N-terminal proteolysis of the a-factor precursor P1 can be accurately reconstituted in vitro using yeast membranes. We show that this activity is dependent on Ste24p and is abolished by mutation of the Ste24p HEXXH metalloprotease motif or by mutation of the a-factor P1 substrate at a residue adjacent to the N-terminal P1 cleavage site. We also demonstrate that N-terminal proteolysis of the P1 a-factor precursor requires Zn(2+) as a co-factor and can be inhibited by the addition of the metalloprotease inhibitor 1,10-orthophenanthroline. Our results are consistent with Ste24p itself being the P1-->P2 a-factor protease or a limiting activator of this activity. Interestingly, we also show that the human Ste24 homolog expressed in yeast can efficiently promote the N-terminal processing of a-factor in vivo and in vitro, thus establishing a-factor as a surrogate substrate in the absence of known human substrates. The results reported here, together with the previously reported in vitro reconstitution of Ste24p-dependent CAAX processing, provide a system for examining the potential bifunctional roles of yeast Ste24p and its homologs.     

Heterologous Expression Studies of Saccharomyces cerevisiae Reveal Two Distinct Trypanosomatid CaaX Protease Activities and Identify Their Potential Targets

The CaaX tetrapeptide motif typically directs three sequential posttranslational modifications, namely, isoprenylation, proteolysis, and carboxyl methylation. In all eukaryotic systems evaluated to date, two CaaX proteases (Rce1 and Ste24/Afc1) have been identified. Although the Trypanosoma brucei genome also encodes two putative CaaX proteases, the lack of detectable T. brucei Ste24 activity in trypanosome cell extracts has suggested that CaaX proteolytic activity within this organism is solely attributed to T. brucei Rce1 (J. R. Gillespie et al., Mol. Biochem. Parasitol. 153:115-124. 2007). In this study, we demonstrate that both T. brucei Rce1 and T. brucei Ste24 are enzymatically active when heterologously expressed in yeast. Using a-factor and GTPase reporters, we demonstrate that T. brucei Rce1 and T. brucei Ste24 possess partially overlapping specificities much like, but not identical to, their fungal and human counterparts. Of interest, a CaaX motif found on a trypanosomal Hsp40 protein was not cleaved by either T. brucei CaaX protease when examined in the context of the yeast a-factor reporter but was cleaved by both in the context of the Hsp40 protein itself when evaluated using an in vitro radiolabeling assay. We further demonstrate that T. brucei Rce1 is sensitive to small molecules previously identified as inhibitors of the yeast and human CaaX proteases and that a subset of these compounds disrupt T. brucei Rce1-dependent localization of our GTPase reporter in yeast. Together, our results suggest the conserved presence of two CaaX proteases in trypanosomatids, identify an Hsp40 protein as a substrate of both T. brucei CaaX proteases, support the potential use of small molecule CaaX protease inhibitors as tools for cell biological studies on the trafficking of CaaX proteins, and provide evidence that protein context influences T. brucei CaaX protease specificity.Certain isoprenylated proteins are synthesized as precursors having a highly degenerate C-terminal tetrapeptide CaaX motif (C, cysteine; a, aliphatic amino acid; X, one of several amino acids). This motif typically directs three posttranslational modifications that include covalent attachment of an isoprenoid lipid to the cysteine residue, followed by endoproteolytic removal of the terminal three residues (i.e., aaX), and lastly, carboxyl methyl esterification of the farnesylated cysteine (49, 50). Relevant examples of proteins subject to the above modifications, also referred to as CaaX proteins, include the Ras and Ras-related GTPases, Gγ subunits, prelamin A, members of the Hsp40 family of chaperones, and fungal mating pheromones.Isoprenylation of CaaX proteins is performed by either the farnesyltransferase (FTase) or the geranylgeranyl transferase I (GGTase I). The particular isoprenoid attached, C15 farnesyl or C20 geranylgeranyl, respectively, depends in part on the sequence of the CaaX motif (8, 26, 31). Proteolysis of isoprenylated intermediates is carried out by the otherwise unrelated Rce1p (Ras converting enzyme 1) and Ste24p (sterile mutant 24) enzymes, collectively referred to as CaaX proteases, which are integral membrane proteins residing within the endoplasmic reticulum (3, 40, 45). Studies to elucidate the specificities of the CaaX proteases have often involved reporters designed from biological substrates (e.g., Ras GTPases) (2, 3, 16, 21, 22, 24, 34). Although these studies suggest that isoprenylated CaaX tetrapeptides alone are sufficient for recognition as a substrate, insufficient evidence exists to assert whether this sequence contains all of the necessary information for substrate specificity. Reporters are typically cleaved by either Rce1p or Ste24p. The Saccharomyces cerevisiae a-factor mating pheromone is a rather unusual biological reporter since it is cleaved by both yeast CaaX proteases. Orthologs of the CaaX proteases from humans, worms, and plants can also cleave a-factor when heterologously expressed in yeast, thereby making a-factor a convenient reporter for comparative analyses of CaaX protease activities (3, 5, 6, 36). Where evaluated using the a-factor reporter, Rce1p and Ste24p display partially overlapping target specificity, and this is an expected property of CaaX proteases in all eukaryotic systems (5, 6, 36, 47). Unlike the isoprenylation and proteolysis steps, carboxyl methyl esterification exclusively relies on a single enzyme, the isoprenylcysteine carboxyl methyltransferase (ICMT) (23, 50). A farnesylated cysteine appears to be the sole recognition determinant of the endoplasmic reticulum-localized ICMT (10, 23, 38).Disruption of the posttranslational modifications associated with CaaX proteins is often perceived as an anticancer strategy because of the prominent role of CaaX proteins in cellular transformation (i.e., the Ras GTPases) (49). To date, the most advanced drug discovery efforts have focused on farnesyltransferase inhibitors (FTIs) (9, 53). Inhibitors of the CaaX proteases and ICMT are also being developed (1, 11, 28, 37, 39, 48). Disrupting CaaX protein modifications has therapeutic application to other diseases as well. The relief of prelamin A toxicity by FTIs is a well-documented example (51). Accumulation of the farnesylated but unproteolysed precursor of lamin A results in a progeroid phenotype in individuals lacking ZmpSte24 proteolytic activity. The treatment of parasitic disease is another area under investigation (13). A number of FTIs have been developed that inhibit protozoan FTases, and in vivo testing is a continued effort (15, 32). Although research is less advanced with respect to CaaX protease and ICMT inhibitors, RNA interference experiments on the bloodstream form of Trypanosoma brucei indicate that CaaX processing enzymes are required for viability and proliferation of the parasite (20).In the present study, we evaluated the enzymatic properties of the trypanosomal CaaX proteases. We establish through the use of a variety of in vivo and in vitro assays that T. brucei Rce1 and T. brucei Ste24 are active when heterologously expressed in S. cerevisiae and have partially overlapping substrate specificities. The assays rely on various reporters, specifically the yeast a-factor mating pheromone, a K-Ras4B-based fluorogenic peptide, a green fluorescent protein (GFP)-GTPase fusion, and a T. brucei Hsp40 protein. All but the GTPase reporter could be effectively cleaved by both T. brucei CaaX proteases. We also demonstrate that the trypanosomal CaaX proteases can be targeted for inhibition by small molecules both in vitro and when heterologously expressed in yeast, suggesting that the trypanosomal CaaX proteases may be attractive drug targets for pharmacological inhibition.     

Modulation of the inhibitor properties of dipeptidyl (acyloxy)methyl ketones toward the CaaX proteases

Dipeptidyl (acyloxy)methyl ketones (AOMKs) have been identified as mechanism-based inhibitors of certain cysteine proteases. These compounds are also inhibitors of the integral membrane proteins Rce1p and Ste24p, which are proteases that independently mediate a cleavage step associated with the maturation of certain isoprenylated proteins. The enzymatic mechanism of Rce1p is ill-defined, whereas Ste24p is a zinc metalloprotease. Rce1p is required for the proper processing of the oncoprotein Ras and is viewed as a potential target for cancer therapy. In this study, we synthesized a small library of dipeptidyl AOMKs to investigate the structural elements that contribute to the inhibitor properties of this class of molecules toward Rce1p and Ste24p. The compounds were evaluated using a fluorescence-based in vitro proteolysis assay. The most potent dipeptidyl AOMKs contained an arginine residue and the identity of the benzoate group strongly influenced potency. A ‘warhead’ free AOMK inhibited Rce1p and Ste24p. The data suggest that the dipeptidyl AOMKs are not mechanism-based inhibitors of Rce1p and Ste24p and corroborate the hypothesis that Rce1p is not a cysteine protease.     

丙型肝炎病毒NS2-3蛋白酶基因在Sf9昆虫细胞中的表达

丙型肝炎病毒(hepatitis C virus,HCV)为单股正链RNA病毒,其基因组长约9.5kb,5'端和3'端各有一个长约345bp和60bp的非编码区,编码区含一个大开放读码框架,编码3 010aa～3 033aa残基的多蛋白前体.     

Human CaaX protease ZMPSTE24 expressed in yeast: Structure and inhibition by HIV protease inhibitors

The function and localization of proteins and peptides containing C‐terminal “CaaX” (Cys‐aliphatic‐aliphatic‐anything) sequence motifs are modulated by post‐translational attachment of isoprenyl groups to the cysteine sulfhydryl, followed by proteolytic cleavage of the aaX amino acids. The zinc metalloprotease ZMPSTE24 is one of two enzymes known to catalyze this cleavage. The only identified target of mammalian ZMPSTE24 is prelamin A, the precursor to the nuclear scaffold protein lamin A. ZMPSTE24 also cleaves prelamin A at a second site 15 residues upstream from the CaaX site. Mutations in ZMPSTE24 result in premature‐aging diseases and inhibition of ZMPSTE24 activity has been reported to be an off‐target effect of HIV protease inhibitors. We report here the expression (in yeast), purification, and crystallization of human ZMPSTE24 allowing determination of the structure to 2.0 Å resolution. Compared to previous lower resolution structures, the enhanced resolution provides: (1) a detailed view of the active site of ZMPSTE24, including water coordinating the catalytic zinc; (2) enhanced visualization of fenestrations providing access from the exterior to the interior cavity of the protein; (3) a view of the C‐terminus extending away from the main body of the protein; (4) localization of ordered lipid and detergent molecules at internal and external surfaces and also projecting through fenestrations; (5) identification of water molecules associated with the surface of the internal cavity. We also used a fluorogenic assay of the activity of purified ZMPSTE24 to demonstrate that HIV protease inhibitors directly inhibit the human enzyme in a manner indicative of a competitive mechanism.     

Topological and Mutational Analysis of Saccharomyces cerevisiae Ste14p, Founding Member of the Isoprenylcysteine Carboxyl Methyltransferase Family

Eukaryotic proteins that terminate in a CaaX motif undergo three processing events: isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. In Saccharomyces cerevisiae, the latter step is mediated by Ste14p, an integral endoplasmic reticulum membrane protein. Ste14p is the founding member of the isoprenylcysteine carboxyl methyltransferase (ICMT) family, whose members share significant sequence homology. Because the physiological substrates of Ste14p, such as Ras and the yeast a-factor precursor, are isoprenylated and reside on the cytosolic side of membranes, the Ste14p residues involved in enzymatic activity are predicted to be cytosolically disposed. In this study, we have investigated the topology of Ste14p by analyzing the protease protection of epitope-tagged versions of Ste14p and the glycosylation status of Ste14p-Suc2p fusions. Our data lead to a topology model in which Ste14p contains six membrane spans, two of which form a helical hairpin. According to this model most of the Ste14p hydrophilic regions are located in the cytosol. We have also generated ste14 mutants by random and site-directed mutagenesis to identify residues of Ste14p that are important for activity. Notably, four of the five loss-of-function mutations arising from random mutagenesis alter residues that are highly conserved among the ICMT family. Finally, we have identified a novel tripartite consensus motif in the C-terminal region of Ste14p. This region is similar among all ICMT family members, two phospholipid methyltransferases, several ergosterol biosynthetic enzymes, and a group of bacterial open reading frames of unknown function. Site-directed and random mutations demonstrate that residues in this region play a critical role in the function of Ste14p.     

Immunolocalization and characterization of two novel proteases in Leishmania donovani: Putative roles in host invasion and parasite development

Two novel intracellular proteases having identical molecular mass (58 kDa) were purified from virulent Indian strain of Leishmania donovani by a combination of aprotinin-agarose affinity chromatography, ion exchange chromatography and finally continuous elution electrophoresis. Both of these proteases migrate in SDS-PAGE as a single homogeneous bands suggesting monomeric nature of these proteases. The enzyme activity of one of the proteases was inhibited by serine protease inhibitor aprotinin and another one was inhibited by metalloprotease inhibitor 1, 10 phenanthroline. The purified enzymes were thus of serine protease (SP-Ld) and metalloprotease (MP-Ld) type. The optimal pH for protease activity is 8.0 and 7.5 for SP-Ld and MP-Ld respectively. The temperature optimum for SP-Ld is 28 °C and for MP-Ld is 37 °C showing their thermostability upto 60 °C. Broad substrate (both natural and synthetic) specificity and the effect of Ca²⁺ upon these enzymes suggested novelty of these proteases. Kinetic data indicate that SP-Ld is of trypsin like as BAPNA appears to be the best substrate and MP-Ld seems to be collagenase type as it degrades azocoll with maximum efficiency. Both immunofluorescence and immune-gold electron microscopy studies revealed that the SP-Ld is localized in the flagellar pocket as well as at the surface of the parasite, whereas MP-Ld is located extensively near the flagellar pocket region. This work also suggests that the uses of anti SP-Ld and anti MP-Ld antibodies are quite significant in interfering with the process of parasite invasion and multiplication respectively. Thus the major role of SP-Ld could be predicted in invasion process as it down regulates the phagocytic activity of macrophages, and MP-Ld appears to play important roles in parasitic development.     

An On-Demand Metalloprotease from Psychro-Tolerant Exiguobacterium undae Su-1, the Activity and Stability of Which Are Controlled by the Ca2+ Concentration

We reported an on-demand type of metalloprotease from Exiguobacterium undae Su-1. Although this species of bacterium is known to inhabit the permafrost, there are no reports on either strong proteases or peptidases. We found that Su-1 protease is superior to commercially available proteases in proteolytic activity in a lower to normal range of temperature (10–50 °C) as well as in rapid inactivation heat-dependently on the Ca²⁺ concentration. These characteristics meet well with the demands from food processing and manufacturing. Biochemical investigations of the purified enzyme and protein structural analysis after gene cloning confirmed that Su-1 protease conserved high identity in its primary sequence with thermophilic proteases of the M4 family. On the other hand, its flexibility was enhanced when one Ca²⁺ binding site was lost and by replacement for proline and isoleucine residues.     

The multispanning membrane protein Ste24p catalyzes CAAX proteolysis and NH2-terminal processing of the yeast a-factor precursor

Saccharomyces cerevisiae Ste24p is a multispanning membrane protein implicated in the CAAX proteolysis step that occurs during biogenesis of the prenylated a-factor mating pheromone. Whether Ste24p acts directly as a CAAX protease or indirectly to activate a downstream protease has not yet been established. In this study, we demonstrate that purified, detergent-solubilized Ste24p directly mediates CAAX proteolysis in a zinc-dependent manner. We also show that Ste24p mediates a separate proteolytic step, the first NH(2)-terminal cleavage in a-factor maturation. These results establish that Ste24p functions both as a bona fide COOH-terminal CAAX protease and as an a-factor NH(2)-terminal protease. Importantly, this study is the first to directly demonstrate that a eukaryotic multispanning membrane protein can possess intrinsic proteolytic activity.     

An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I

A method was developed to eliminate the proteases contaminating commercial DNase I, which can cause degradation of target protein during the purification process. Bio Basic DNase stock solution (in Tris–HCl buffer [pH 8.0] containing 5 mM CaCl₂) was first incubated at 50 °C to generate autolysis of proteases and zymogens, leading to a significant reduction in protease activity while preserving DNase activity. The residual protease activity was completely inhibited by further incubation with 2 mM PMSF (phenylmethylsulfonyl fluoride) or 2× S8830 inhibitor cocktail. This approach could be readily applicable to eliminate the protease activity in any DNase products or during the preparation of commercial DNase.     

A new intracellular serine protease inhibitor expressed in the rat pituitary gland complexes with granzyme B

We have cloned a novel serpin (raPIT5a) from a rat pituitary cDNA library which is structurally related to members of the ovalbumin subfamily of serine protease inhibitors. This new cDNA encodes a 374-amino acid protein, designated raPIT5a. raPIT5a was expressed in specific cells in the intermediate and anterior lobes of the pituitary. Recombinant raPIT5a was not secreted suggesting raPIT5a functions to inhibit intracellular proteases. Recombinant raPIT5a formed an SDS-stable complex with human granzyme B, a serine protease which induces apoptosis by activating members of the caspase enzyme family. These data suggest raPIT5a may have a role in regulating granzyme B or related enzymes and apoptosis in the pituitary gland.     

Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease

Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q black triangle down A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30 degrees C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days.     

Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease

A cDNA clone, MT-c, encoding metalloprotease was isolated from snake (Agkistrodon halys brevicadus) venom gland cDNA library. Deduced amino acid sequence indicated that MT-c is composed of a signal sequence, amino-terminal propeptide, a central metalloprotease domain, and a Lys-Gly-Asp (KGD) disintegrin domain. The partial cDNA encoding metalloprotease and disintegrin domain was subcloned and expressed in E. coli. The expressed MT-c protein was purified and successfully refolded into functional form retaining the enzyme activity. Analyses of the purified recombinant protease activity revealed that the enzyme hydrolyzes extracellular matrix proteins including type I gelatin, type IV and type V collagen, while type I, II, III collagens and fibronectin were insensitive to the proteolytic digestion. The recombinant enzyme was also able to degrade fibrinogen by specifically cleaving A alpha chain of the protein.     

ADAM 12, a disintegrin metalloprotease, interacts with insulin-like growth factor-binding protein-3

Insulin-like growth factor-binding protein (IGFBP)-3 binds the insulin-like growth factors with high affinity and modulates their actions. Proteolytic cleavage of IGFBP-3 may regulate insulin-like growth factor bioavailability. IGFBP-3 is extensively degraded in serum during pregnancy; however, as yet the pregnancy-specific protease, or proteases, have not been identified. We utilized a yeast two-hybrid assay and a human placental cDNA library to investigate IGFBP-3-interacting proteins. A disintegrin and metalloprotease-12 (ADAM 12), a member of a family of metalloprotease disintegrins that is highly expressed in placental tissue, was identified as interacting with IGFBP-3. This interaction involved the cysteine-rich domain of ADAM 12. Unlike other members of this family of disintegrin metalloproteases that are membrane proteins, ADAM 12 exists as an alternatively spliced soluble secreted protein. To verify the interaction between ADAM 12 and IGFBP-3, an expression construct containing an ADAM 12-S cDNA was transfected into COS-1 cells. Co-precipitation was observed when conditioned medium was analyzed by immunoprecipitation with an antibody against either ADAM 12 or IGFBP-3 followed by Western blotting with anti-IGFBP-3 or anti-ADAM 12. Although minimal proteolysis of IGFBP-3 was observed in conditioned medium from control cells, this was increased approximately 4-fold in conditioned medium from ADAM 12-S-transfected cells. Recombinant ADAM 12-S partially purified from conditioned medium on a heparin-Sepharose column also proteolyzed IGFBP-3. The degradation pattern was similar to that seen with pregnancy serum, and the presence of ADAM 12-S in serum during pregnancy was confirmed. The data suggest that ADAM 12-S has IGFBP-3 protease activity, and it may contribute to the IGFBP-3 protease activity present in pregnancy serum.