Mario R. W. Ehlers

Ace revisited: A new target for structure-based drug design

Current-generation angiotensin-converting enzyme (ACE) inhibitors are widely used for cardiovascular diseases, including high blood pressure, heart failure, heart attack and kidney failure, and have combined annual sales in excess of US

Interactions between Mycobacterium tuberculosis and host cells: are mycobacterial sugars the key?

6 billion. However, the use of these ACE inhibitors, which were developed in the late 1970s and early 1980s, is hampered by common side effects. Moreover, we now know that ACE actually consists of two parts (called the N- and C-domains) that have different functions. Therefore, the design of specific domain-selective ACE inhibitors is expected to produce next-generation drugs that might be safer and more effective. Here we discuss the structural features of current inhibitors and outline how next-generation ACE inhibitors could be designed by using the three-dimensional molecular structure of human testis ACE. The ACE structure provides a unique opportunity for rational drug design, based on a combination of in silico modelling using existing inhibitors as scaffolds and iterative lead optimization to drive the synthetic chemistry.

Identification of N-Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form

Mycobacterium tuberculosis has evolved successful strategies to invade and persist within macrophages. Intimate pathogen-macrophage contacts dictate receptor choice and probably specify the intracellular fate of these microorganisms. Binding to specific receptors, such as complement receptor type 3, could provide an advantage. These interactions appear to involve surface polysaccharides and glycolipids.

Deglycosylation, processing and crystallization of human testis angiotensin-converting enzyme.

The sites of glycosylation of Chinese hamster ovary cell expressed testicular angiotensin-converting enzyme (tACE) have been determined by matrix-assisted laser desorption ionization/time of flight/mass spectrometry of peptides generated by proteolytic and cyanogen bromide digestion. Two of the seven potential N-linked glycosylation sites, Asn90 and Asn109, were found to be fully glycosylated by analysis of peptides before and after treatment with a series of glycosidases and with endoproteinase Asp-N. The mass spectra of the glycopeptides exhibit characteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type. This structural information was used to demonstrate that three other sites, Asn155, Asn337, and Asn586, are partially glycosylated, whereas Asn72 appears to be fully glycosylated. The only potential site that was not modified is Asn620. Sequence analysis of tryptic peptides obtained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a counterpart in tACE. Comparison of the two proteins reveals a pattern in which amino-terminal N-linked sites are preferred. The functional significance of glycosylation was examined with a tACE mutant lacking the O-glycan-rich first amino-terminal 36 residues and truncated at Ser625. When expressed in the presence of the α-glucosidase I inhibitor N-butyldeoxynojirimycin and treated with endoglycosidase H to remove all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a good candidate for crystallographic studies.

The unique N-terminal sequence of testis angiotensin-converting enzyme is heavily O-glycosylated and unessential for activity or stability

Angiotensin I-converting enzyme (ACE) is a highly glycosylated type I integral membrane protein. A series of underglycosylated testicular ACE (tACE) glycoforms, lacking between one and five N-linked glycosylation sites, were used to assess the role of glycosylation in tACE processing, crystallization and enzyme activity. Whereas underglycosylated glycoforms showed differences in expression and processing, their kinetic parameters were similar to that of native tACE. N-glycosylation of Asn-72 or Asn-109 was necessary and sufficient for the production of enzymically active tACE but glycosylation of Asn-90 alone resulted in rapid intracellular degradation. All mutants showed similar levels of phorbol ester stimulation and were solubilized at the same juxtamembrane cleavage site as the native enzyme. Two mutants, tACEDelta36-g1234 and -g13, were successfully crystallized, diffracting to 2.8 and 3.0 A resolution respectively. Furthermore, a truncated, soluble tACE (tACEDelta36NJ), expressed in the presence of the glucosidase-I inhibitor N -butyldeoxynojirimycin, retained the activity of the native enzyme and yielded crystals belonging to the orthorhombic P2(1)2(1)2(1) space group (cell dimensions, a=56.47 A, b=84.90 A, c=133.99 A, alpha=90 degrees, beta=90 degrees and gamma=90 degrees ). These crystals diffracted to 2.0 A resolution. Thus underglycosylated human tACE mutants, lacking O-linked oligosaccharides and most N-linked oligosaccharides or with only simple N-linked oligosaccharides attached throughout the molecule, are suitable for X-ray diffraction studies.

Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages

The testis-specific isozyme of angiotensin-converting enzyme (ACE) is identical, from residue 68 to the C terminus, to the second half or C-terminal domain of somatic ACE. However, the first 67 residues, comprising the signal peptide and a Ser-/Thr-rich 36-residue sequence that constitutes the N terminus of mature testis ACE, are unique. We have expressed a mutant human testis ACE lacking this 36-residue N-terminal sequence and find that compared to the wild-type protein the mutant is 15 kDa smaller due to the loss of greater than 90% of all O-linked sugars, but that it retains full enzymatic activity and is stable in culture. Heavy O-glycosylation is a property of testis ACE that is not shared by the somatic enzyme and is attributable to this unique sequence.

The mycosins of Mycobacterium tuberculosis H37Rv: a family of subtilisin-like serine proteases

BackgroundExported proteases are commonly associated with virulence in bacterial pathogens, yet there is a paucity of information regarding their role in Mycobacterium tuberculosis. There are five genes (mycP1-5) present within the genome of Mycobacterium tuberculosis H37Rv that encode a family of secreted, subtilisin-like serine proteases (the mycosins). The gene mycP1 (encoding mycosin-1) was found to be situated 3700 bp (four ORFs) from the RD1 deletion region in the genome of the attenuated vaccine strain M. bovis BCG (bacille de Calmette et Guérin) and was selected for further analyses due to the absence of expression in this organism.ResultsFull-length, 50 kDa mycosin-1 was observed in M. tuberculosis cellular lysates, whereas lower-molecular-weight species were detected in culture filtrates. A similar lower-molecular-weight species was also observed during growth in macrophages. Mycosin-1 was localized to the membrane and cell wall fractions in M. tuberculosis by Western blotting, and to the cell envelope by electron microscopy. Furthermore, M. tuberculosis culture filtrates were shown to contain a proteolytic activity inhibited by mixed serine/cysteine protease inhibitors and activated by Ca2+, features typical of the subtilisins.ConclusionsMycosin-1 is an extracellular protein that is membrane- and cell wall-associated, and is shed into the culture supernatant. The protein is expressed after infection of macrophages and is subjected to proteolytic processing. Although proteolytically active mycosin-1 could not be generated recombinantly, serine protease activity containing features typical of the subtilisins was detected in M. tuberculosis culture filtrates.

Roles of the juxtamembrane and extracellular domains of angiotensin-converting enzyme in ectodomain shedding

There is little information regarding the role of proteolysis in Mycobacterium tuberculosis and no studies on the potential involvement of proteases in the pathogenesis of tuberculosis. We identified five M. tuberculosis genes (mycP1-5) that encode a family of serine proteases (mycosins-1 to 5), ranging from 36 to 47% identity. Each protein contains a catalytic triad (Asp, His, Ser) within highly conserved sequences, typical of proteases of the subtilisin family. These genes are also present in M. bovis BCG and other virulent mycobacteria, but only one homologue (mycP3) was detected in M. smegmatis. The mycosins have N-terminal signal sequences and C-terminal transmembrane anchors, and the localisation of the mycosins to the membrane/cell wall was verified by Western blot analysis of heterologously expressed proteins in cellular fractions of M. smegmatis. In M. tuberculosis, all the mycosins were expressed constitutively during growth in broth. Mycosins-2 and 3 were also expressed constitutively in M. bovis BCG, but no expression of mycosin-1 was detected. Mycosin-2 was modified by cleavage in all three mycobacterial species. The multiplicity and constitutive expression of these proteins suggests that they have an important role in the biology of M. tuberculosis.

Purification and characterization of recombinant human testis angiotensin-converting enzyme expressed in chinese hamster ovary cells

Angiotensin-converting enzyme (ACE) is one of a growing number of integral membrane proteins that is shed from the cell surface through proteolytic cleavage by a secretase. To investigate the requirements for ectodomain shedding, we replaced the glycosylphosphatidylinositol addition sequence in membrane dipeptidase (MDP) - a membrane protein that is not shed - with the juxtamembrane stalk, transmembrane (TM) and cytosolic domains of ACE. The resulting construct, MDP-STM(ACE), was targeted to the cell surface in a glycosylated and enzymically active form, and was shed into the medium. The site of cleavage in MDP-STM(ACE) was identified by MS as the Arg(374)-Ser(375) bond, corresponding to the Arg(1203)-Ser(1204) secretase cleavage site in somatic ACE. The release of MDP-STM(ACE) and ACE from the cells was inhibited in an identical manner by batimastat and two other hydroxamic acid-based zinc metallosecretase inhibitors. In contrast, a construct lacking the juxtamembrane stalk, MDP-TM(ACE), although expressed at the cell surface in an enzymically active form, was not shed, implying that the juxtamembrane stalk is the critical determinant of shedding. However, an additional construct, ACEDeltaC, in which the N-terminal domain of somatic ACE was fused to the stalk, TM and cytosolic domains, was also not shed, despite the presence of a cleavable stalk, implying that in contrast with the C-terminal domain, the N-terminal domain lacks a signal required for shedding. These data are discussed in the context of two classes of secretases that differ in their requirements for recognition of substrate proteins.

Immune-modulating effects of alpha-1 antitrypsin

Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: p omega-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which full-length and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mg/liter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either p omega-ACE or pLEN-ACE-VII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) approximately 100 kDa. The native and recombinant proteins cross-reacted equally with anti-human kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investigating structure-function relationships in this enzyme.