Neprilysin 2: a novel messenger peptide-inactivating metalloprotease

Neprilysin 2 is a recently identified glycoprotein displaying the highest degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 family of zinc-dependent metalloproteases. Whereas neprilysin has been shown to be involved in the inactivation of endogenous messenger peptides, like enkephalins and tachykinins, the true physiological functions of neprilysin 2 remain unknown.     

Endothelin-3-converting enzyme activity of the KEL1 and KEL6 phenotypes of the Kell blood group system

The Kell blood group protein is a metalloendopeptidase that preferentially cleaves a Trp(21)-Ile(22) bond of big endothelin-3 producing bioactive endothelin-3. Kell is a polymorphic protein, and 25 different phenotypes, because of point mutations resulting in single amino acid substitutions, have been described. It was recently reported that a recombinant form of KEL1 (K, K1) phenotype, expressed in K562 and HEK293 cells, had no endothelin-3-converting activity, in contrast to the common KEL2 (k, K2) phenotype. We demonstrate that KEL1 red blood cells and also a soluble recombinant form of KEL1 protein (s-Kell KEL1) have similar enzymatic activity as the common Kell phenotype. In addition we show that KEL6 red blood cells, which are more prevalent in persons of African heritage than in Caucasians also have endothelin-3-converting enzyme activity and that the recombinant soluble form of KEL6 protein (s-Kell KEL6) has similar K(m) values as the wild-type.     

Bioinformatic analysis of the neprilysin (M13) family of peptidases reveals complex evolutionary and functional relationships

Background  

The neprilysin (M13) family of endopeptidases are zinc-metalloenzymes, the majority of which are type II integral membrane proteins. The best characterised of this family is neprilysin, which has important roles in inactivating signalling peptides involved in modulating neuronal activity, blood pressure and the immune system. Other family members include the endothelin converting enzymes (ECE-1 and ECE-2), which are responsible for the final step in the synthesis of potent vasoconstrictor endothelins. The ECEs, as well as neprilysin, are considered valuable therapeutic targets for treating cardiovascular disease. Other members of the M13 family have not been functionally characterised, but are also likely to have biological roles regulating peptide signalling. The recent sequencing of animal genomes has greatly increased the number of M13 family members in protein databases, information which can be used to reveal evolutionary relationships and to gain insight into conserved biological roles.     

Kell blood group antigens are part of a 93,000-dalton red cell membrane protein

Monospecific Kell blood group antibodies, of either human alloimmune or mouse monoclonal origin, react with a single surface-exposed protein of 93,000 daltons. Chymotryptic peptide maps of the 93,000-dalton protein isolated by antibodies of two different specificities (anti-K7 or anti-K14) indicate that Kell epitopes reside on the same protein. Kell protein is similar in size to band 3 protein but differs markedly in its tryptic and chymotryptic peptide maps, indicating that they are different proteins. In addition, sheep antibody to human band 3 does not react with Kell protein. Rabbit antibody to Kell protein reacts, by Western immunoblotting, with membrane proteins from Kell antigen positive red blood cells but not from those of a Ko (Kell null) cell. In intact red cells only a small portion of the Kell protein is available to lactoperoxidase-catalyzed iodination. Under nonreducing conditions Kell antigen is isolated not only as a 93,000-dalton protein but also as larger protein complexes ranging in size from above 200,000 to 115,000 daltons. Treatment of red cells with iodoacetamide, prior to isolation of Kell protein, reduces the amount of the very large complexes, but Kell protein occurs both as 115,000- and 93,000-dalton proteins.     

The mouse Kell blood group gene (Kel): cDNA sequence, genomic organization, expression, and enzymatic function

The human Kell blood group system is important in transfusion medicine, since Kell is a polymorphic protein and some of its antigens can cause severe reactions if mismatched blood is transfused, while maternal alloimmunization may lead to fetal and neonatal anemia. In humans, Kell is an Mr 93,000 type II membrane glycoprotein with endothelin-3-converting enzyme activity that is linked by a single disulfide bond to another protein, XK, that spans the membrane ten times. An absence of XK leads to clinical symptoms termed the McLeod syndrome. We determined the cDNA sequence of the mouse Kell homologue, the organization of the gene, expression of the protein and its enzymatic function on red cells. Comparison of human and mouse Kell cDNA showed 80% nucleotide and 74% amino acid sequence identity. Notable differences are that the mouse Kell protein has eight probable N-linked carbohydrate side chains, compared to five for human Kell, and that the mouse homologue has one more extracellular cysteine than human Kell protein. The mouse Kell gene (Kel), like its human counterpart, is similarly organized into 19 exons. Kel was located to proximal Chromosome 6. Northern blot analysis showed high expression in spleen and weaker levels in testis and heart. Western blot analysis of red cell membrane proteins demonstrated that mouse Kell glycoprotein has an apparent Mr of 110,000 and, on removal of N-linked sugars, 80,000. As in human red cells, Kell is disulfide-linked to XK and mouse red cells have endothelin-3-converting enzyme activity.     

The ultimate tryptophan residue of neprilysin 2 is not involved in protein maturation and enzymatic activity

Modeling the three-dimensional structure of neprilysin 2 (NEP2) using the crystal structure of neprilysin as template revealed that their active sites share many common features, though slight differences therein cannot completely account for their specific pharmacological profiles. Recent evidence also suggest that residues outside the active site can play crucial functions in the maturation and enzymatic activity of these metalloproteases. To further explore the functions of amino acids in the acquisition and maintenance of the NEP2 structure, site-directed mutagenesis of conserved residues involved in the enzymatic activity of ECE-1 was performed. In particular, the ultimate tryptophan residue of ECE-1 was recently shown to be important in its activation. This residue was thus mutated in the secreted isoform of NEP2, as were proline residues located in its vicinity. Expression of these mutants in AtT20 cells and study of their secretion and catalytic activities shows that while the ultimate tryptophan residue of the NEP2 sequence is not essential to its proper and activity, structural changes in its vicinity can have a severe impact on the maturation processes involved in the activation of NEP2.     

Computer modeling and nanosecond simulation of the enzyme–substrate complex of the common lymphoblastic leukemia antigen (neprilysin) indicates shared residues at the primary specificity pocket (S1') with matrix metalloproteases

The common acute lymphoblastic leukemia antigen (neprilysin, CD10, neutral endopeptidase 24.11) is a member of the neprilysin family, and projects functions in signaling pathways in pathophysiological processes such as cancer, Alzheimer's disease and hypertension. Given its pathophysiological importance, an investigation of the natural substrate specificity of this metalloprotease is presented here through the application of enzyme-substrate modeling and molecular dynamics simulations. The results show that the substrate modeled, LATAC downward arrow FG, satisfies a complementary backbone H-bonding with Ala543-Tyr545, thereby suggested to be the putative substrate-binding beta-sheet, analogously to matrix metalloproteases. The modeling further suggests that phenylalanine at the P1' position (substrate) is directed in the same fashion as the synthetic inhibitor of the reference crystal structure and that this enzyme does not bind the P3'/P4' positions of a substrate, as other metalloproteases do. After a specific comparison with one member of the matrix metalloproteases, MMP-3, a common conserved valine residue at the primary S1' subsite was found to be shared between these two otherwise different proteases. These results may prove useful for selective drug design for neprilysin, and lay a foundation for future subsite analysis for other members of the neprilysin family.     

Distribution of the rol gene encoding the regulator of lipopolysaccharide O-chain length in Escherichia coli and its influence on the expression of group I capsular K antigens.

The rol (cld) gene encodes a protein involved in the expression of lipopolysaccharides in some members of the family Enterobacteriaceae. Rol interacts with one or more components of Rfc-dependent O-antigen biosynthetic complexes to regulate the chain length of lipopolysaccharide O antigens. The Rfc-Rol-dependent pathway for O-antigen synthesis is found in strains with heteropolysaccharide O antigens, and, consistent with this association, rol-homologous sequences were detected in chromosomal DNAs from 17 different serotypes with heteropolysaccharide O antigens. Homopolymer O antigens are synthesized by a pathway that does not involve either Rfc or Rol. It was therefore unexpected when a survey of Escherichia coli strains possessing mannose homopolymer O8 and O9 antigens showed that some strains contained rol. All 11 rol-positive strains coexpressed a group IB capsular K antigen with the O8 or O9 antigen. In contrast, 12 rol-negative strains all produced group IA K antigens in addition to the homopolymer O antigen. Previous research from this and other laboratories has shown that portions of the group I K antigens are attached to lipopolysaccharide lipid A-core, in a form that we have designated K(LPS). By constructing a hybrid strain with a deep rough rfa defect, it was shown that the K40 (group IB) K(LPS) antigen exists primarily as long chains. However, a significant amount of K40 antigen was surface expressed in a lipid A-core-independent pathway. The typical chain length distribution of the K40 antigen was altered by introduction of multicopy rol, suggesting that the K40 group IB K antigen is equivalent to a Rol-dependent O antigen. The prototype K30 (group IA) K antigen is expressed as short oligosaccharides (primarily single repeat units) in K(LPS), as well as a high-molecular-weight lipid A-core-independent form. Introduction of multicopy rol into the K30 strain generated a novel modal pattern of K(LPS) with longer polysaccharide chains. Collectively, these results suggested that group IA K(LPS) is also synthesized by a Rol-dependent pathway and that the typically short oligosaccharide K(LPS) results from the absence of Rol activity in these strains.     

Tissue transglutaminase is a multifunctional BH3-only protein

Tissue transglutaminase (TG2) protein accumulates to high levels in cells during early stages of apoptosis both in vivo and in vitro. The analysis of the TG2 primary sequence showed the presence of an eight amino acid domain, sharing 70% identity with the Bcl-2 family BH3 domain. Cell-permeable peptides, mimicking the domain sequence, were able to induce Bax conformational change and translocation to mitochondria, mitochondrial depolarization, release of cytochrome c, and cell death. Moreover, we found that the TG2-BH3 peptides as well as TG2 itself were able to interact with the pro-apoptotic Bcl-2 family member Bax, but not with anti-apoptotic members Bcl-2 and Bcl-X(L). Mutants in the TG2-BH3 domain failed to sensitize cells toward apoptosis. In TG2-overexpressing cells about half of the protein is localized on the outer mitochondrial membrane where, upon cell death induction, it cross-links many protein substrates including Bax. TG2 is the first member of a new subgroup of multifunctional BH3-only proteins showing a large mass size (80 kDa) and enzymatic activity.     

The amyloid precursor-like protein 2 associates with the major histocompatibility complex class I molecule K(d)

Amyloid precursor-like protein 2 (APLP2) is a member of a protein family related to the amyloid precursor protein, which is implicated in Alzheimer's disease. Little is known about the physiological function of this protein family. The adenovirus E3/19K protein binds to major histocompatibility complex (MHC) class I antigens in the endoplasmic reticulum, thereby preventing their transport to the cell surface. In cells coexpressing E3/19K and the MHC K(d) molecule, K(d) is associated with E3/19K and two cellular protein species with masses of 100 and 110 kDa, termed p100/110. Interestingly, p100/110 are released from the complex upon the addition of K(d)-binding peptides, suggesting a role for these proteins in peptide transfer to MHC molecules. Here we demonstrate by microsequencing, reactivity with APLP2-specific antibodies, and comparison of biochemical parameters that p100/110 is identical to human APLP2. We further show that the APLP2/K(d) association does not require the physical presence of E3/19K. Thus, APLP2 exhibits an intrinsic affinity for the MHC K(d) molecule. Similar to the binding of MHC molecules to the transporter associated with antigen processing, complex formation between APLP2 and K(d) strictly depends upon the presence of beta(2)-microglobulin. Conditions that prolong the residency of K(d) in the endoplasmic reticulum lead to a profound increase of the association and a drastic reduction of APLP2 transport. Therefore, this unexpected interplay between these unrelated molecules may have implications for both MHC antigen and APLP2 function.     

The E3/19K protein of adenovirus type 2 binds to the domains of histocompatibility antigens required for CTL recognition

The E3/19K protein of human adenovirus type 2 binds to HLA class I antigens and blocks their terminal glycosylation and cell surface expression. The nature of this interaction is non-covalent and involves neither disulfide bridges between the two molecules nor their carbohydrates. The murine H-2 Kd antigen associates with the E3/19K protein in a similar fashion to human HLA antigens whereas the allelic product H-2 Kk does not. Hybrid genes between the Kd and Kk alleles were constructed and their products were expressed in embryonic kidney cells together with the E3/19K protein. This allowed us to identify the alpha 1 and alpha 2 domains as the essential structures of the histocompatibility antigens for binding the viral protein. Interestingly, these domains are also crucial for T cell recognition. The implications for the evolution of adenoviruses and their ability to cause persistent infections are discussed.     

The structure, expression, and properties of additional members of the protein kinase C family

In rat brain three members of the protein kinase C family encoded by cDNAs termed delta, epsilon, and zeta were newly identified by molecular cloning and sequence analysis. The new members have a common structure that is closely related to but clearly distinct from the four members of the family previously isolated having alpha-, beta I-, beta II-, and gamma-sequences, although the zeta-cDNA available at present does not appear to contain a complete reading frame for protein kinase C. The delta-, epsilon-, and zeta-cDNAs all encode a characteristic cysteine-rich sequence and protein kinase domain sequence, both of which are highly homologous among the protein kinase C family. However, the new members lack one of the conserved regions that is present in alpha-, beta I-, beta II-, and gamma-sequences. An additional cDNA clone termed epsilon' was isolated, which is identical with epsilon-cDNA except for a short sequence at the 5'-terminal end region. The two members having delta- and epsilon-sequences were expressed in COS 7 cells, and partially purified and characterized. The enzymes having delta- and epsilon-sequences depend on phospholipid and diacylglycerol for the enzymatic activity, but their properties slightly differ from the previously known members of protein kinase C. Northern blot analysis suggests that the new members of protein kinase C exist in the brain and some other tissues.     

Intracellular behavior of rabies virus matrix protein (M) is determined by the viral glycoprotein (G).

To investigate the nature and intracellular behavior of the matrix (M) protein of an avirulent strain (HEP-Flury) of rabies virus, we cloned and sequenced the cDNA of the protein. Using expression vectors pZIP-NeoSV(X)1 and pCDM8, the cDNA was transfected to animal cells (BHK-21 and COS-7) with or without coexpression of viral glycoprotein (G). When M protein alone was expressed in the cells, it displayed homogeneous distribution in the whole cell including the nucleus. In contrast, coexpression with G protein resulted in the abolishment of nuclear distribution of M antigen, and both of the antigens displayed a colocalized distribution in the cell, especially at the cellular membrane as seen in the virus-infected cells, while the distribution of G antigen was not affected by coexpressed M antigen. Immunoprecipitation studies revealed that M protein was coprecipitated with G protein by anti-G antibody, and vice versa, although cross-linking with dithiobis(succinimidyl propionate) was necessary for coprecipitation because of their easier dissociation in the presence of sodium deoxycholate. These results suggest that M protein intimately associates with G protein, which may affect or regulate the behavior (e.g., intracellular localization) of M protein. Studies with deletion mutants of M protein indicate that an internal region around the amino acids from 115 to 151 is essential for the M protein to preserve its binding ability to G protein.     

Laeverin/aminopeptidase Q, a novel bestatin-sensitive leucine aminopeptidase belonging to the M1 family of aminopeptidases

Laeverin/aminopeptidase Q (APQ) is a cell surface protein specifically expressed on human embryo-derived extravillous trophoblasts that invades the uterus during placentation. The cDNA cloning of Laeverin/APQ revealed that the sequence encodes a protein with 990 amino acid residues, and Laeverin/APQ contains the HEXXHX(18)E gluzincin motif, which is characteristic of the M1 family of aminopeptidases, although the exopeptidase motif of the family, GAMEN, is uniquely substituted for the HAMEN sequence. In this study, we expressed a recombinant human Laeverin/APQ using a baculovirus expression system, purified to homogeneity, and characterized its enzymatic properties. It was found that Laeverin/APQ had a broad substrate specificity toward synthetic substrate, although it showed a preference for Leu-4-methylcoumaryl-7-amide. Searching natural substrates, we found that Laeverin/APQ was able to cleave the N-terminal amino acid of several peptides such as angiotensin III, kisspeptin-10, and endokinin C, which are abundantly expressed in the placenta. In contrast to the case with other M1 aminopeptidases, bestatin inhibited the aminopeptidase activity of Laeverin/APQ much more effectively than other known aminopeptidase inhibitors. These results indicate that Laeverin/APQ is a novel bestatin-sensitive leucine aminopeptidase and suggest that the enzyme plays important roles in human placentation by regulating biological activity of key peptides at the embryo-maternal interface.     

Intracellular assembly of Kell and XK blood group proteins.

Kell, a 93 kDa type II membrane glycoprotein, and XK, a 444 amino acid multi-pass membrane protein, are blood group proteins that exist as a disulfide-bonded complex on human red cells. The mechanism of Kell/XK assembly was studied in transfected COS cells co-expressing Kell and XK proteins. Time course studies combined with endonuclease-H treatment and cell fractionation showed that Kell and XK are assembled in the endoplasmic reticulum. At later times the Kell component of the complex was not cleaved by endonuclease-H, indicating N-linked oligosaccharide processing and transport of the complex to a Golgi and/or a post-Golgi cell fraction. Surface-labeling of transfected COS cells, expressing both Kell and XK, demonstrated that the Kell/XK complex travels to the plasma membrane. XK expressed in the absence of Kell was also transported to the cell surface indicating that linkage of Kell and XK is not obligatory for cell surface expression.     

A three-dimensional model of the neprilysin 2 active site based on the X-ray structure of neprilysin. Identification of residues involved in substrate hydrolysis and inhibitor binding of neprilysin 2

Neprilysin 2 (NEP2), a recently identified member of the M13 subfamily of metalloproteases, shares the highest degree of homology with the prototypical member of the family neprilysin. Whereas the study of the in vitro enzymatic activity of NEP2 shows that it resembles that of NEP as it cleaves the same substrates often at the same amide bonds and binds the same inhibitory compounds albeit with different potencies, its physiological role remains elusive because of the lack of selective inhibitors. To aid in the design of these novel compounds and better understand the different inhibitory patterns of NEP and NEP2, the x-ray structure of NEP was used as a template to build a model of the NEP2 active site. The results of our modeling suggest that the overall structure of NEP2 closely resembles that of NEP. The model of the active site reveals a 97% sequence identity with that of NEP with differences located within the S'(2) subsite of NEP2 where Ser(133) and Leu(739) replace two glycine residues in NEP. To validate the proposed model, site-directed mutagenesis was performed on a series of residues of NEP2, mutants expressed in AtT20 cells, and their ability to bind various substrates and inhibitory compounds was tested. The results confirm the involvement of the conserved Arg(131) and Asn(567) in substrate binding and catalytic activity of NEP2 and further show that the modifications in its S'(2) pocket, particularly the presence therein of Leu(739), account for a number of differences in inhibitor binding between NEP and NEP2.     

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn.

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.     

Structural characterization and chromosomal localization of the MAGE-E1 gene

Genes of the melanoma-associated antigen (MAGE) family are characterized by the expression of tumor antigens on a malignant melanoma recognized by autologous cytolytic T lymphocytes. We have previously identified novel members of the MAGE gene family expressed in human glioma and named them MAGE-E1a-c. In the present study, we have revealed the genomic structure of MAGE-E1 by sequence analysis of a human chromosome bacterial artificial chromosome clone containing the MAGE-E1 gene. The MAGE-E1 gene is composed of 13 exons, and three of these (exon 2, exon 3 and exon 12) are alternatively spliced in each variant (E1a-c). The open reading frame encoding the MAGE-E1 peptides initiates in exon 2 and ends in exon 13. We have also demonstrated that the MAGE-E1 gene is located in Xp11 through the analysis of radiation hybrid panels. The genomic structure of MAGE-E1 is markedly similar to that of MAGE-D and its chromosomal locus is also identical to that of MAGE-D, but these features contrast with those of other MAGEs. These results suggest that MAGE-D and -E1 may be evolutionarily distant from other members of the MAGE family, and the two may be ancestral genes for the others.