The function of the milk-clotting enzymes bovine and camel chymosin studied by a fluorescence resonance energy transfer assay.

Enzymatic coagulation of bovine milk can be divided in 2 steps: an enzymatic step, in which the Phe105-Met106 bond of the milk protein bovine κ-casein is cleaved, and an aggregation step. The aspartic peptidases bovine and camel chymosin (EC 3.4.23.4) are typically used to catalyze the enzymatic step. The most commonly used method to study chymosin activity is the relative milk-clotting activity test that measures the end point of the enzymatic and aggregation step. This method showed that camel chymosin has a 2-fold higher milk-clotting activity toward bovine milk than bovine chymosin. To enable a study of the enzymatic step independent of the aggregation step, a fluorescence resonance energy transfer assay has been developed using a peptide substrate derived from the 98-108 sequence of bovine κ-casein. This assay and Michaelis-Menten kinetics were employed to determine the enzymatic activity of camel and bovine chymosin under milk clotting-like conditions (pH 6.65, ionic strength 80 mM). The results obtained show that the catalytic efficiency of camel chymosin is 3-fold higher than bovine chymosin. The substrate affinity and catalytic activity of bovine and camel chymosin increase at lower pH (6.00 and 5.50). The glycosylation of bovine and camel chymosin did not affect binding of the fluorescence resonance energy transfer substrate, but doubly glycosylated camel chymosin seems to have slightly higher catalytic efficiency. In the characterization of the enzymes, the developed assay is easier and faster to use than the traditionally used relative milk-clotting activity test method.

Selective inhibition of ADAM12 catalytic activity through engineering of tissue inhibitor of metalloproteinase 2 (TIMP-2).

The disintegrin and metalloprotease ADAM12 has important functions in normal physiology as well as in diseases, such as cancer. Little is known about how ADAM12 confers its pro-tumorigenic effect; however, its proteolytic capacity is probably a key component. Thus selective inhibition of ADAM12 activity may be of great value therapeutically and as an investigative tool to elucidate its mechanisms of action. We have previously reported the inhibitory profile of TIMPs (tissue inhibitor of metalloproteinases) against ADAM12, demonstrating in addition to TIMP-3, a unique ADAM-inhibitory activity of TIMP-2. These findings strongly suggest that it is feasible to design a TIMP mutant selectively inhibiting ADAM12. With this purpose, we characterized the molecular determinants of the ADAM12-TIMP complex formation as compared with known molecular requirements for TIMP-mediated inhibition of ADAM17/TACE (tumour necrosis factor alpha-converting enzyme). Kinetic analysis using a fluorescent peptide substrate demonstrated that the molecular interactions of N-TIMPs (N-terminal domains of TIMPs) with ADAM12 and TACE are for the most part comparable, yet revealed strikingly unique features of TIMP-mediated ADAM12 inhibition. Intriguingly, we found that removal of the AB-loop in N-TIMP-2, which is known to impair its interaction with TACE, resulted in increased affinity to ADAM12. Importantly, using a cell-based epidermal growth factor-shedding assay, we demonstrated for the first time an inhibitory activity of TIMPs against the transmembrane ADAM12-L (full-length ADAM12), verifying the distinctive inhibitory abilities of N-TIMP-2 and engineered N-TIMP-2 mutants in a cellular environment. Taken together, our findings support the idea that a distinctive ADAM12 inhibitor with future therapeutic potential can be designed.

Expression, purification and insights into structure and folding of the ADAM22 pro domain.

The ADAMs (a disintegrin and metalloproteases) are an important class of enzymes in the regulation of human disease. The pro domains of ADAMs are responsible for the latency and secretion of mature enzymes. Unlike other metzincins, ADAM pro domains remain bound to the mature enzyme after secretion. To understand the functions of human ADAM pro domains and to determine three-dimensional structures, we have screened promising targets for expression and purification properties when using Escherichia coli as the host. The pro domain of ADAM22 (ADAM22-P) expressed in E. coli was folded, as determined by CD and NMR spectroscopy. An ADAM22-P fragment encoding residues 26-199 could be expressed in high amounts, remained soluble above 1 mM, and was suitable for structural studies by NMR spectroscopy. CD spectroscopy and predictions suggest that the secondary structure in ADAM22-P consists of beta-strands. Furthermore, our data indicate that the pro domains of ADAMs are expressed as two subdomains. The most N-terminal subdomain (ADAM22-P(N)) was found to be susceptible to proteolysis and was required for folding stability of the second subdomain (ADAM22-P(C)).

Culture independent PCR: an alternative enzyme discovery strategy.

Degenerate primers were designed for use in a culture-independent PCR screening of DNA from composite fungal communities, inhabiting residues of corn stovers and leaves. According to similarity searches and alignments amplified clone sequences affiliated with glycosyl hydrolase family 7 and glycosyl hydrolase family 45 though significant sequence divergence was observed. Glycosyl hydrolases from families 7 and 45 play a crucial role in biomass conversion to fuel ethanol. Research in this renewable energy source has two objectives: (i) To contribute to development of a renewable alternative to world's limited crude fossil oil reserves and (ii) to reduce air pollution. Amplification with 18S rDNA-specific primers revealed species within the ascomycetous orders Sordariales and Hypocreales as well as basidiomycetous order Agaricales to be present in these communities. Our study documents the value of culture-independent PCR in microbial diversity studies and could add to development of a new enzyme screening technology.

Catalytic properties of ADAM12 and its domain deletion mutants.

Human ADAM12 (a disintegrin and metalloproteinase) is a multidomain zinc metalloproteinase expressed at high levels during development and in human tumors. ADAM12 exists as two splice variants: a classical type 1 membrane-anchored form (ADAM12-L) and a secreted splice variant (ADAM12-S) consisting of pro, catalytic, disintegrin, cysteine-rich, and EGF domains. Here we present a novel activity of recombinant ADAM12-S and its domain deletion mutants on S-carboxymethylated transferrin (Cm-Tf). Cleavage of Cm-Tf occurred at multiple sites, and N-terminal sequencing showed that the enzyme exhibits restricted specificity but a consensus sequence could not be defined as its subsite requirements are promiscuous. Kinetic analysis revealed that the noncatalytic C-terminal domains are important regulators of Cm-Tf activity and that ADAM12-PC consisting of the pro domain and catalytic domain is the most active on this substrate. It was also observed that NaCl inhibits ADAM12. Among the tissue inhibitors of metalloproteinases (TIMP) examined, the N-terminal domain of TIMP-3 (N-TIMP-3) inhibits ADAM12-S and ADAM12-PC with low nanomolar Ki(app) values while TIMP-2 inhibits them with a slightly lower affinity (9-44 nM). However, TIMP-1 is a much weaker inhibitor. N-TIMP-3 variants that lack MMP inhibitory activity but retained the ability to inhibit ADAM17/TACE failed to inhibit ADAM12. These results indicate unique enzymatic properties of ADAM12 among the members of the ADAM family of metalloproteinases.

Heparan sulfate regulates ADAM12 through a molecular switch mechanism.

The disintegrin and metalloproteases (ADAMs) are emerging as therapeutic targets in human disease, but specific drug design is hampered by potential redundancy. Unlike other metzincins, ADAM prodomains remain bound to the mature enzyme to regulate activity. Here ADAM12, a protease that promotes tumor progression and chondrocyte proliferation in osteoarthritic cartilage, is shown to possess a prodomain/catalytic domain cationic molecular switch, regulated by exogenous heparan sulfate and heparin but also endogenous cell surface proteoglycans and the polyanion, calcium pentosan polysulfate. Sheddase functions of ADAM12 are regulated by the switch, as are proteolytic functions in placental tissue and sera of pregnant women. Moreover, human heparanase, an enzyme also linked to tumorigenesis, can promote ADAM12 sheddase activity at the cell surface through cleavage of the inhibitory heparan sulfate. These data present a novel concept that might allow targeting of ADAM12 and suggest that other ADAMs may have specific regulatory activity embedded in their prodomain and catalytic domain structures.

ADAM12 is a four-leafed clover: the excised prodomain remains bound to the mature enzyme.

The ADAMs (a disintegrin and metalloprotease) comprise a family of multidomain proteins with metalloprotease, cell adhesion, and signaling activities. Human ADAM12, which is implicated in diseases such as cancer, is expressed in two splice forms, the transmembrane ADAM12-L and the shorter and soluble ADAM12-S. ADAM12 is synthesized as a zymogen with the prodomain keeping the metalloprotease inactive through a cysteine-switch mechanism. Maturation and activation of the protease involves the cleavage of the prodomain in the trans-Golgi or possibly at the cell surface by a furin-peptidase. The aim of the present study was to determine the fate of the prodomain following furin cleavage. Here we demonstrate that, following cleavage of the human ADAM12-S prodomain in the trans-Golgi by a furin-peptidase, the prodomain remains non-covalently associated with the mature molecule. Accordingly, both the 68-kDa mature form of ADAM12-S and the 25-kDa prodomain could be detected using domain-specific antisera in immunoprecipitation and Western blot analyses of human serum ADAM12 and purified recombinant human ADAM12. Using electron microscopy after negative staining we have furthermore obtained the first visualization of a full-length ADAM molecule, human ADAM12-S, and report that it appears to be a compact clover composed of four globular domains, one of which is the prodomain. Finally, our data demonstrate that the presence of the metalloprotease domain appears to be sufficient for the prodomain to remain associated with the mature ADAM12-S. Thus, we conclude that the prodomain of human ADAM12-S is an integral domain of the mature molecule and as such might have specific biological functions in the extracellular space.

Beringian paleoecology inferred from permafrost-preserved fungal DNA.

The diversity of fungi in permanently frozen soil from northeastern Siberia was studied by culture-independent PCR amplification of diverse environmental 18S rRNA genes. Elaborate protocols to avoid contamination during drilling, sampling, and amplification were used. A broad diversity of eukaryotic DNA sequences that were 510 bp long, including sequences of various fungi, plants, and invertebrates, could be obtained reproducibly from samples that were up to 300,000 to 400,000 years old. The sequences revealed that ancient fungal communities included a diversity of cold-adapted yeasts, dark-pigmented fungi, plant-parasitic fungi, and lichen mycobionts. DNA traces of tree-associated macrofungi in a modern tundra sample indicated that there was a shift in fungal diversity following the last ice age and supported recent results showing that there was a severe change in the plant composition in northeastern Siberia during this period. Interestingly, DNA sequences with high homology to sequences of coprophilic and keratinophilic fungi indicated that feces, hair, skin, and nails could have been sources of ancient megafauna DNA recently reported to be present in small amounts of Siberian permafrost sediments.