Crystal structure of N-acyl-D-glucosamine 2-epimerase from porcine kidney at 2.0 A resolution

The X-ray crystallographic structure of N-acyl-d-glucosamine 2-epimerase (AGE) from porcine kidney, which has been identified to be a renin-binding protein (RnBP), was determined by the multiple isomorphous replacement method and refined at 2.0 A resolution with a final R-factor of 16.9 % for 15 to 2.0 A resolution data. The refined structure of AGE comprised 804 amino acid residues (one dimer) and 145 water molecules. The dimer of AGE had an asymmetric unit with approximate dimensions 46 Ax48 Ax96 A. The AGE monomer is composed of an alpha(6)/alpha(6)-barrel, the structure of which is found in glucoamylase and cellulase. One side of the AGE alpha(6)/alpha(6)-barrel structure comprises long loops containing five short beta-sheets, and contributes to the formation of a deep cleft shaped like a funnel. The putative active-site pocket and a possible binding site for the substrate N-acetyl-d-glucosamine (GlcNAc) were found in the cleft. The other side of the alpha(6)/alpha(6)-barrel comprises short loops and contributes to the dimer formation. At the dimer interface, which is composed of the short loops and alpha-helices of the subunits, five strong ion-pair interactions were observed, which play a major role in the dimer assembly. This completely ruled out the previously accepted hypothesis that the formation of the RnBP homodimer and RnBP-renin heterodimer requires the leucine zipper motif present in RnBP.     

Genetic and molecular properties of human and rat renin-binding proteins with reference to the function of the leucine zipper motif.

The presence of a leucine zipper motif was recognized in the deduced amino acid sequences of human and rat renin-binding proteins (RnBPs) on cloning and sequence analysis of the RnBP cDNAs. The in vitro synthesized RnBPs, with the respective cDNAs, formed heterodimers with porcine renin and homodimers. On comparison of these properties with those of porcine RnBP, the leucine zipper motif was suggested to be a functional domain common to animal RnBPs. In addition to the motif, a hydrophobic domain adjacent to the motif and 10 cysteine residues were also well conserved in the three RnBPs. Moreover, about 85% of their amino acid sequences were identical. The RnBP mRNAs were expressed in the kidneys as the same size of 1.5-kb and the genes are suggested to exist as single copies in the genomes. Despite the high similarities in genetic and molecular properties, the molecular weights of human and rat RnBPs were 43,000, which is 1,000 larger than that of porcine RnBP. The immunoreactivities of human and rat RnBPs toward anti-porcine RnBP antiserum were 88 and 8% that of porcine RnBP, respectively, and the affinities of the two RnBPs for porcine renin were remarkably less than that of porcine RnBP. Moreover, the human and rat RnBP homodimers were partly dissociated under the conditions under which porcine RnBP existed as a dimer. These results indicate distinct differences in the molecular properties among the three RnBPs, in spite of their being highly similar structurally and functionally.     

Purification and characterization of renin binding protein (RnBP) from porcine kidney

Renin binding protein (RnBP) was purified from porcine kidney using pepstatin affinity column chromatography, DEAE-Sepharose column chromatography, gel filtration on Ultrogel-AcA 34, aminohexyl-Sepharose 4B column chromatography, and high performance liquid chromatography (HPLC) on TSK-gel G-3000 SW. The purified preparation was homogeneous as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, polyacrylamide disc gel electrophoresis and isoelectric focusing on polyacrylamide gel. The isoelectric point was at pH 4.85, and the apparent molecular weight of RnBP was estimated to be 42,000 by SDS-polyacrylamide gel electrophoresis. The preparation did not show any renin activity and was stable for 30 min at 37 degrees C between pH 5.0 and 9.0 or on storage for 4 weeks at 4 degrees C or -80 degrees C. The activity of renin was greatly inhibited by RnBP. From the kinetic analysis of the inhibition we roughly estimated the dissociation constant between renin and RnBP to be about 0.2 nM, assuming that the stoichiometry in the complex, i.e., high molecular weight (HMW) renin, is one to one, and that the complex is inactive. The inhibitory activity of RnBP was lost by acidification at pH 3.0 and the activity of renin was restored. The purified RnBP formed a single precipitin line with the antiserum prepared with the purified HMW renin as antigen, which is RnBP-renin complex (Takahashi, S., et al. (1983) J. Biochem. 93, 265-274), and this line fused with one of the two precipitin lines formed between HMW renin and anti-HMW renin antiserum. The other of the two lines was between renin and anti-HMW renin antiserum. The purified preparation was thus identified as RnBP. The HMW renin was reconstituted with the purified RnBP and renin, and the apparent molecular weight of the reconstituted specimen was estimated to be 60,000 by gel filtration on Ultrogel AcA 44.     

Renin inhibits N-acetyl-D-glucosamine 2-epimerase (renin-binding protein)

Renin-binding protein (RnBP) is a highly specific renin inhibitor first isolated from porcine kidney. Our recent studies demonstrated that the human RnBP is the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353]. We have developed a new assay method for GlcNAc 2-epimerase activity using a system of N-acyl-D-hexosamine oxidase coupled with peroxidase and employed this method to study the effects of renin on GlcNAc 2-epimerase activity. The recombinant human (rh) RnBP existed as a dimer and its GlcNAc 2-epimerase activity was strongly inhibited by the purified renin concomitant with the formation of RnBP-renin heterodimer, so-called high molecular weight (HMW) renin. The renin activity was also inhibited by rhRnBP in a dose-dependent manner. These results indicate that renin is an inhibitor of GlcNAc 2-epimerase, and the renin-RnBP heterodimer HMW renin is an inactive form of both renin and GlcNAc 2-epimerase activities.     

A study on renin binding protein (RnBP) in the human kidney

We purified, from human kidney, a protein that reacts with rabbit anti-porcine kidney renin binding protein (RnBP) antiserum by trapping with porcine kidney renin. The purified preparation showed a single protein peak on gel filtration by high performance liquid chromatography (HPLC) and two protein bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The latter two kinds of protein were identified as the porcine renin and human kidney protein from their electrophoretic mobilities and reactivity toward rabbit anti-porcine kidney renin and RnBP antisera. The molecular weights of the purified preparation and the human kidney protein were estimated to be 56,000 by HPLC and 43,000 by SDS-PAGE, respectively. The specific activity of porcine renin in the purified preparation was 8.6 mg angiotensin I per mg of protein per h at 37 degrees C and pH 6.5. This specific activity was about one-fifth that of free porcine renin. Therefore, it is suggested from the reactivity toward the anti-porcine RnBP antiserum and inhibitory action toward porcine renin that the human kidney protein is RnBP and that the human RnBP is purified as a complex with porcine renin.     

Effects of nucleotides on the interaction of renin with GlcNAc 2-epimerase (renin binding protein, RnBP)

Renin binding protein (RnBP), a cellular renin inhibitor, was identified as an enzyme, GlcNAc 2-epimerase. Recombinant RnBP inhibited porcine renin activity in a dose dependent manner. However, the inhibition was neutralized by nucleotides, such as ATP, dATP, dGTP, dCTP or dTTP. Moreover, ATP inhibited the formation of hetero-complex of renin with RnBP, called high molecular weight (HMW) renin. On the other hand, N-ethylmaleimide (NEM), a SH-alkylating reagent inhibited the GlcNAc 2-epimerase activity concomitant with the decaying of the dimer to the monomer of the enzyme. The inhibition was modulated in the presence of ATP. These results indicate that nucleotides stabilize the dimeric form RnBP (GlcNAc 2-epimerase) and inhibited the formation of the renin-RnBP hetero complex, HMW renin.     

Homodimerization via a leucine zipper motif is required for enzymatic activity of quiescent cell proline dipeptidase

Quiescent cell proline dipeptidase (QPP) is an intracellular serine protease that is also secreted upon cellular activation. This enzyme cleaves N-terminal Xaa-Pro dipeptides from proteins, an unusual substrate specificity shared with dipeptidyl peptidase IV (CD26/DPPIV). QPP is a 58-kDa protein that elutes as a 120-130-kDa species from gel filtration, indicating that it forms a homodimer. We analyzed this dimerization with in vivo co-immunoprecipitation assays. The amino acid sequence of QPP revealed a putative leucine zipper motif, and mutational analyses indicated that this leucine zipper is required for homodimerization. The leucine zipper mutants showed a complete lack of enzymatic activity, suggesting that homodimerization is important for QPP function. On the other hand, an enzyme active site mutant retained its ability to homodimerize. These data are the first to demonstrate a role for a leucine zipper motif in a proteolytic enzyme and suggest that leucine zipper motifs play a role in mediating dimerization of a diverse array of proteins.     

The divergent domains of the NEFA and nucleobindin proteins are derived from an EF-hand ancestor

The human protein NEFA (DNA binding, EF-hand, Acidic region) has previously been isolated from a KM3 cell line and immunolocalized on the plasma membrane, in the cytoplasma, and in the culture medium. Sequence analysis of a cDNA clone encoding NEFA identified a hydrophilic domain, two EF-hands, and a leucine zipper at the C- terminus. These characters are shared with nucleobindin (Nuc). In this paper we have further characterized NEFA and probed its evolutionary origins. Circular dichroism (CD) spectra of recombinant NEFA indicated a helical content of 51% and showed that the EF-hands are capable of binding Ca2+. Experiments with recombinant NEFA and synthesized peptides revealed that the leucine zipper cannot form a homodimer. The leucine zipper may allow heterodimer formation of NEFA and an unknown protein. Phylogenetic analyses suggest that this protein is derived from a four-domain EF-hand ancestor with subsequent duplications and fusions. The leucine zipper and putative DNA-binding domains of NEFA have evolved secondarily from existing EF-hand sequences. These analyses provide insights into how complex proteins may originate and trace the precursor of NEFA to the common ancestor of eukaryotes.      

Modulation of active renin secretion by renin-binding protein (RnBP) in mouse pituitary AtT-20 cells transfected with human renin and RnBP cDNAs.

To investigate the role of renin-binding protein (RnBP) in renin metabolism, RnBP expression plasmid, which was constructed to express human RnBP under the control of mouse mammary tumor virus long terminal repeat, was transfected into mouse pituitary AtT-20 cells together with the expression plasmid encoding human renin. The transfectant secreted prorenin and active renin, whereas RnBP was expressed only in the presence of dexamethasone and without secretion into the medium. The secretion of active renin was stimulated by forskolin, and the stimulation was repressed by dexamethasone. The secretion of prorenin, however, was insensitive to forskolin irrespective of the presence or absence of dexamethasone. Moreover, the forskolin-stimulated release of active renin was hardly repressed by dexamethasone in AtT-20 cells transfected with the renin expression plasmid and a selectable plasmid pMAMneo. Coexistence of RnBP and renin mRNAs in human Wilms' tumor G-401 cells was shown by means of polymerase chain reaction of respective cDNAs from the cells. These results suggest that RnBP modulates the release of active renin in renin-producing cells.     

Molecular cloning and sequence analysis of a cDNA encoding a porcine kidney renin-binding protein

A complementary DNA encoding a renin-binding protein (RnBP) has been isolated from a porcine kidney cDNA library by immunological screening of in vitro translation products from the cDNAs. Analysis of the nucleotide sequence of the clone revealed a 1,342-nucleotide sequence with a 5'-terminal untranslated region of 52 nucleotides, an open reading frame of 1,206 nucleotides that encodes 402 amino acids, and a 3'-terminal untranslated region of 84 nucleotides that contains the polyadenylation signal sequence, AATAAA. The predicted amino acid sequence contains no hydrophobic amino-terminal sequence and does not show significant homology to those of other identified proteins. The in vitro translated RnBP was found to have the same molecular weight, 42,000, as that of the purified RnBP from porcine kidney on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and it formed a complex with renin purified from porcine kidney, which indicates that the cDNA encodes a functional RnBP without a propeptide sequence. The RnBP cDNA probe hybridized to a 1.5-kilobase mRNA in kidney, liver, adrenal, and pituitary glands, the amount being much greater in kidney than in the other tissues. Southern blot analysis showed the presence of a unique gene for RnBP in the porcine genome.     

Tissue-specific regulation of renin-binding protein gene expression in rats.

Rat gene for renin-binding protein (RnBP) was shown to be expressed in the kidney, adrenal gland, brain, lung, spleen, ovary, testis, and heart. On sodium depletion and captopril administration, the rat showed a marked increase in the adrenal RnBP mRNA level and a slight decrease in the kidney RnBP mRNA level. In two-kidney, one clip hypertensive rats, the RnBP mRNA levels of the clipped and contralateral kidneys were unchanged and also its adrenal mRNA level was maintained at the control level. The recombinant rat RnBP was synthesized in Escherichia coli cells and purified to apparent homogeneity. The RnBP existed as a homodimer and formed a heterodimer with rat renin to inhibit renin activity extensively. Intravenous injection of the RnBP into rats resulted in a rapid and strong inhibition of plasma renin activity, which persisted at least for 2 h. These results suggest that the expression of RnBP gene in the kidney and adrenal gland is regulated independently, and the function of RnBP is related to electrolyte homeostasis, probably through the interaction with renin.     

Purification of high molecular weight (HMW) renin from porcine kidney and direct evidence that the HMW renin is a complex of renin with renin binding protein (RnBP)

The high molecular weight (HMW) renin was purified from porcine kidney by a procedure involving extraction with a buffer system containing protease inhibitors, ammonium sulfate fractionation, pepstatin-aminohexyl-Sepharose 4B column chromatography, gel filtration on Ultrogel AcA 44 and aminohexyl-Sepharose 4B column chromatography. The resulting preparation showed a single band on isoelectric focusing, exhibiting an isoelectric point at pH 5.25, and was stable on storage at -80 degrees C for 4 months. The specific activity was 3.97 mg of angiotensin I formed/mg of protein per h at 37 degrees C and at pH 6.5 with porcine angiotensinogen as the substrate. When the HMW renin was exposed to acid, renin activity increased by about 5-fold and the free form of fully active renin was recovered from the acidified HMW renin, leaving an insoluble aggregate of protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the HMW renin showed two protein bands, of which one was identified as renin from the electrophoretic mobility and the other was the protein, assigned as renin binding protein (RnBP), that was insolubilized by acidification. The purified HMW renin is a complex of renin with RnBP, and the molecular weights of RnBP and renin in the HMW renin were estimated to be 39,000 and 32,000, respectively, by gel permeation liquid chromatography in 6 M guanidine-HCl. A modified rapid method for purification of renin is also presented.     

Activity of the human centrosomal kinase, Nek2, depends on an unusual leucine zipper dimerization motif.

Nek2 is a human cell cycle-regulated kinase that is structurally related to the mitotic regulator, NIMA, of Aspergillus nidulans. Localization studies have shown that Nek2 is a core component of the centrosome, the microtubule organizing center of the cell, and functional approaches suggest a possible role for Nek2 in centrosome separation at the G2/M transition. Here, we have investigated the importance of an unusual leucine zipper coiled-coil motif present in the C-terminal noncatalytic domain of the Nek2 kinase. Glycerol gradient centrifugation indicated that endogenous Nek2 is present in HeLa cells as a salt-resistant 6 S complex, the predicted size of a Nek2 homodimer. Recombinant Nek2 overexpressed in insect cells also formed a 6 S complex, whereas a Nek2 mutant specifically lacking the leucine zipper motif was monomeric. Using yeast two-hybrid interaction analyses and coprecipitation assays, we found that Nek2 can indeed form homodimers both in vivo and in vitro and that this dimerization specifically required the leucine zipper motif. Moreover, deletion of the leucine zipper prevented a trans-autophosphorylation reaction on the C-terminal domain of Nek2 and strongly reduced Nek2 kinase activity on exogenous substrates. Finally, we emphasize that the Nek2 leucine zipper described here differs from classical leucine zippers in that it exhibits a radically different arrangement of hydrophobic and charged amino acids. Thus, this study reveals not only an important mechanism for the regulation of the Nek2 kinase but, furthermore, highlights an unusual organization of a leucine zipper dimerization motif.     

Dual DNA recognition codes of a short peptide derived from the basic leucine zipper protein EmBP1

Sequence-specific DNA binding of short peptide dimers derived from a plant basic leucine zipper protein EmBP1 was studied. A homodimer of the EmBP1 basic region peptide recognized a palindromic DNA sequence, and a heterodimer of EmBP1 and GCN4 basic region peptides targets a non-palindromic DNA sequence when a beta-cyclodextrin/adamantane complex is utilized as a dimerization domain. A homodimer of the EmBP1 basic region peptide binds the native EmBP1 binding 5'-GCCACGTGGC-3' and the native GCN4 binding 5'-ATGACGTCAT-3' sequences with almost equal affinity in the alpha-helical conformation, indicating that the basic region of EmBP1 by itself has a dual recognition codes for the DNA sequences. The GCN4 basic region peptide binds 5'-ATGAC-3' in the alpha-helical conformation, but it neither shows affinity nor helix formation with 5'-GCCAC-3'. Because native EmBP1 forms 100 times more stable complex with 5'-GCCACGTGGC-3' over 5'-ATGACGTCAT-3', our results suggest that the sequence-selectivity of native EmBP1 is dictated by the structure of leucine zipper dimerization domain including the hinge region spanning between the basic region and the leucine zipper.     

Biosynthesis of a renin binding protein

The biosynthesis of a porcine renin binding protein (RnBP), which specifically binds to renin and forms an inactive high molecular weight renin, was investigated. mRNAs from various porcine tissues were used to investigate in vitro protein synthesis. The kidney mRNA directed the synthesis of a high level of RnBP, whereas the liver, adrenal and pituitary gland mRNAs gave as low but significant level of it. The in vitro synthesized RnBP as well as the immunologically detected RnBP synthesized in vivo had the same molecular weight, 42,000, as that of the purified protein. Moreover, both the human and rat kidney mRNAs directed the synthesis of this protein identified with an anti-porcine RnBP antibody. These results strongly indicate that RnBP, present in various mammalian species, is synthesized in renin-producing tissues as the mature size and undergoes binding with renin without proteolytic processing.     

Golgi retention of human protein NEFA is mediated by its N-terminal Leu/Ile-rich region.

The subcellular localization of the human Ca(2+)-binding EF-hand/leucine zipper protein NEFA was studied in HeLa cells by immunofluorescence microscopy. Double immunostaining using mouse anti-NEFA monoclonal antibody 1H8D12 and rabbit anti-ERD2 polyclonal antibody proved that NEFA is localized in the Golgi apparatus. The result was confirmed by the expression of NEFA-green fluorescent protein (GFP) fusion protein in the Golgi in the same cell line. Cycloheximide treatment proved NEFA to be a Golgi-resident protein. Seven NEFA deletion mutants were constructed to ascertain the peptide region relevant for Golgi retention. The expression of each NEFA-GFP variant was detected by fluorescence microscopy and immunoblotting. Only the DeltaN mutant, lacking the N-terminal Leu/Ile-rich region, failed to be retained in the Golgi after cycloheximide treatment. The other six deletion mutants in which either the basic region, the complete EF-hand pair domain, the two EF-hand motifs separately, the leucine zipper and the leucine zipper plus the C-terminal region is deleted, were localized to the Golgi. The peptide sequence within the Leu/Ile-rich region is discussed as a novel Golgi retention motif.     

Mutational analysis of the leucine zipper-like motif of the human immunodeficiency virus type 1 envelope transmembrane glycoprotein.

The N-terminal region of the envelope (env) transmembrane protein of human immunodeficiency virus type 1 (HIV-1) has a leucine zipper-like motif. This highly conserved zipper motif, which consists of a heptad repeat of leucine or isoleucine residues, has been suggested to play a role in HIV-1 env glycoprotein oligomerization. This hypothesis was tested by replacing the highly conserved leucine or isoleucine residues in the zipper motif with a strong alpha-helix breaker, proline. We report here that such substitutions did not abolish the ability of env protein to form oligomers, indicating that this highly conserved zipper motif does not have a crucial role in env protein oligomerization. However, the mutant viruses all showed impaired infectivity, suggesting that this conserved zipper motif can have an important role in the virus life cycle.     

LIP19, a basic region leucine zipper protein, is a Fos-like molecular switch in the cold signaling of rice plants

The rice low-temperature-induced lip19 gene encodes a 148-amino-acid basic region/leucine zipper (bZIP) protein, termed LIP19. In this study we characterized LIP19 and showed that it lacks the usual ability of bZIP proteins to homodimerize and to bind DNA, as does the Fos protein in mammals. Using a yeast two-hybrid system, the cDNA clones whose products interact with LIP19 were screened. This search revealed a clone termed OsOBF1 (Oryza sativa OBF1) that encodes a new bZIP protein (OsOBF1). This protein forms a homodimer and binds to the hexamer motif sequence (5'-ACGTCA-3'). The protein-protein interaction in homo- and hetero-combinations between LIP19 and OsOBF1 was confirmed in vitro and in planta. LIP19 and OsOBF1 most likely interact with each other more strongly than OsOBF1 interacts with itself, and the resulting heterodimer binds to the C/G hybrid sequence but not to the hexamer sequence. Whereas the expression patterns of lip19 and OsOBF1 in response to low temperatures were totally opposite, the locations of their expression were almost identical. Based upon the presented data, we propose a model describing the low-temperature signal switching mediated by LIP19 in rice.