Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3.

Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.

Novel synthetic peptide derived from Porphyromonas gingivalis Lys-gingipain detects IgG-mediated host response in periodontitis.

Porphyromonas gingivalis is a keystone pathogen in periodontitis. Analysis of the immunogenicity of its virulence factors may provide insight into the host response to this infection. The Kgp12 (IEDB Epitope ID 763561), an epitope of Lys-gingipain (Kgp) virulence factor from P. gingivalis ATCC 33277, elicits an immunoglobulin G (IgG) immunoreactivity with low cross-reactivity and, therefore, more specificity. The aim of the present study was to determine in silico the localization of Kgp12 within the protein and to evaluate the IgG host response to this novel Kgp peptide through its capacity to differentiate individuals with different periodontal status. Sera of 71 volunteers were tested by indirect ELISA to detect the IgG immunoreactivity specific to Kgp12, as well as to the protein HmuY and to the sonicated total extract of P. gingivalis ATCC33277, both used as gold standard. The participants had no systemic disease and were classified according to periodontal clinical parameters to comparison, firstly, into periodontitis (P) and without periodontitis (WP) groups and, secondly, into periodontitis (P), gingivitis (G) and clinically health (CH) ones. All the antigens tested, Kgp12 (p = 0.02), HmuY (p = 0.00) and P. gingivalis extract (p = 0.03), could differentiate P from WP groups considering IgG serum levels. P group also had higher IgG levels specific to Kgp12 (p = 0.03), HmuY (p < 0.01) and P. gingivalis extract (p = 0.01) when compared to G group. We conclude that the Kgp12 synthetic peptide was useful to detect the IgG-mediated host response signaling that it is a promising epitope to analyze the immunogenicity of P. gingivalis.

Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function.

Porphyromonas gingivalis, a keystone pathogen of chronic periodontitis, uses ferric uptake regulator homolog (PgFur) to regulate production of virulence factors. This study aimed to characterize PgFur protein in regard to its structure-function relationship. We experimentally identified the 5' mRNA sequence encoding the 171-amino-acid-long PgFur protein in the A7436 strain and examined this PgFur version as a full-length protein. PgFur protein did not bind to the canonical Escherichia coli Fur box, but the wild-type phenotype of the mutant Δpgfur strain was restored partially when expression of the ecfur gene was induced from the native pgfur promoter. The full-length PgFur protein contained one zinc atom per protein monomer, but did not bind iron, manganese, or heme. Single cysteine substitutions of CXXC motifs resulted in phenotypes similar to the mutant Δpgfur strain. The modified proteins were produced in E. coli at significantly lower levels, were highly unstable, and did not bind zinc. The pgfur gene was expressed at the highest levels in bacteria cultured for 24 h in the absence of iron and heme or at higher levels in bacteria cultured for 10 h in the presence of protoporphyrin IX source. No influence of high availability of Fe2+, Zn2+, or Mn2+ on pgfur gene expression was observed. Two chromosomal mutant strains producing protein lacking 4 (pgfurΔ4aa) or 13 (pgfurΔ13aa) C-terminal amino acid residues were examined in regard to importance of the C-terminal lysine-rich region. The pgfurΔ13aa strain showed a phenotype typical for the mutant Δpgfur strain, but both the wild-type PgFur protein and its truncated version bound zinc with similar ability. The Δpgfur mutant strain produced higher amounts of HmuY protein compared with the wild-type strain, suggesting compromised regulation of its expression. Potential PgFur ligands, Fe2+, Mn2+, Zn2+, PPIX, or serum components, did not influence HmuY production in the Δpgfur mutant strain. The mutant pgfurΔ4aa and pgfurΔ13aa strains exhibited affected HmuY protein production. PgFur, regardless of the presence of the C-terminal lysine-rich region, bound to the hmu operon promoter. Our data suggest that cooperation of PgFur with partners/cofactors and/or protein/DNA modifications would be required to accomplish its role played in an in vivo multilayer regulatory network.

Production of interferon-gamma, interleukin-6, and interleukin-1ß by human peripheral blood mononuclear cells stimulated with novel lys-gingipain synthetic peptides.

BACKGROUND: Periodontitis is a progressive inflammatory process, and its pathogenesis is related to the presence of a dysbiotic subgingival biofilm that elicits the immune response. Porphyromonas gingivalis is a keystone pathogen, and its Lys-gingipain (Kgp) virulence factor is involved in the pathogen-host interaction through the production of cytokines by host cells, but the specific mechanisms of this interaction have not been elucidated. The present study evaluated the in vitro production of interferon-gamma (IFN-γ), interleukin (IL)-6, and IL-1ß cytokines in response to antigenic stimulation of peripheral blood mononuclear cells (PBMCs) with novel Kgp synthetic peptides. METHODS: Our previous in silico study predicted 16 immunogenic peptides from Kgp protein. Nine peptides derived from different regions of the protein were chemically synthesized. The synthetic peptides Kgp12, 17, and 18 were selected based on the immunoglobulin G immunoreactivity in the serum of patients with periodontitis (P) and individuals without periodontitis (WP), and they were used in in vitro stimulation of PBMC derived from groups P and WP. Enzyme-linked immunosorbent assay and microsphere-based flow cytometric assay were used to verify the levels of the cytokines produced in PBMC cultures after 48 hours. RESULTS: Kgp12, 17, and 18 peptides induced lower production of IFN-γ. Kgp12 induced higher levels of IFN-γ in WP than in P individuals. Kgp12 induced higher production of IL-6 and IL-1ß compared with the other stimuli. CONCLUSION: The novel Kgp synthetic peptides tested herein are immunogenic peptides (epitopes) since they induced the production of cytokines by PBMC and therefore may be useful tools in evaluating the pathogen-host interaction.

SLC35A5 Protein-A Golgi Complex Member with Putative Nucleotide Sugar Transport Activity.

Solute carrier family 35 member A5 (SLC35A5) is a member of the SLC35A protein subfamily comprising nucleotide sugar transporters. However, the function of SLC35A5 is yet to be experimentally determined. In this study, we inactivated the SLC35A5 gene in the HepG2 cell line to study a potential role of this protein in glycosylation. Introduced modification affected neither N- nor O-glycans. There was also no influence of the gene knock-out on glycolipid synthesis. However, inactivation of the SLC35A5 gene caused a slight increase in the level of chondroitin sulfate proteoglycans. Moreover, inactivation of the SLC35A5 gene resulted in the decrease of the uridine diphosphate (UDP)-glucuronic acid, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine Golgi uptake, with no influence on the UDP-galactose transport activity. Further studies demonstrated that SLC35A5 localized exclusively to the Golgi apparatus. Careful insight into the protein sequence revealed that the C-terminus of this protein is extremely acidic and contains distinctive motifs, namely DXEE, DXD, and DXXD. Our studies show that the C-terminus is directed toward the cytosol. We also demonstrated that SLC35A5 formed homomers, as well as heteromers with other members of the SLC35A protein subfamily. In conclusion, the SLC35A5 protein might be a Golgi-resident multiprotein complex member engaged in nucleotide sugar transport.

Lysine at position 329 within a C-terminal dilysine motif is crucial for the ER localization of human SLC35B4.

SLC35B4 belongs to the solute carrier 35 (SLC35) family whose best-characterized members display a nucleotide sugar transporting activity. Using an experimental model of HepG2 cells and indirect immunofluorescent staining, we verified that SLC35B4 was localized to the endoplasmic reticulum (ER). We demonstrated that dilysine motif, especially lysine at position 329, is crucial for the ER localization of this protein in human cells and therefore one should use protein C-tagging with caution. To verify the importance of the protein in glycoconjugates synthesis, we generated SLC35B4-deficient HepG2 cell line using CRISPR-Cas9 approach. Our data showed that knock-out of the SLC35B4 gene does not affect major UDP-Xyl- and UDP-GlcNAc-dependent glycosylation pathways.

Antimicrobial activity of stable hemiaminals against Porphyromonas gingivalis.

Porphyromonas gingivalis is a major etiologic agent and a key pathogen responsible for the development and progression of chronic periodontitis. Controlling the number of periodontal pathogens is one of the primary actions for maintaining oral health; therefore, active compounds with a capacity to exert antimicrobial activity have received considerable attention as they may represent potential new therapeutic agents for the treatment of chronic periodontitis. Heterocyclic compounds possessing 1,2,4- or 1,2,3-triazoles are known for several biological activities, including antibacterial properties. Among them are stable hemiaminals which can be obtained in reaction between nitrobenzaldehyde derivatives and 4-amino-1,2,4-triazole or 4-amino-3,5-dimethyl-1,2,4-triazole. In this study, we selected two relatively stable hemiaminals: (2,4-dinitrophenyl)(4H-1,2,4-triazole-4-ylamino)methanol (24DNTAM) and (2,4-dinitrophenyl)(4H-3,5-dimethyl-1,2,4-triazole-4-ylamino)methanol (24DNDMTAM). Both compounds showed promising anti-P. gingivalis activity, higher against ATCC 33277 strain as compared to A7436 strain. The lowest hemiaminal concentration inhibiting visible planktonic bacterial growth under high-iron/heme conditions was ∼0.06 mg/ml, and the lowest hemiaminal concentration showing killing of bacteria was ∼0.25 mg/ml. Antimicrobial activity was also observed against P. gingivalis grown on blood agar plates. Slightly higher antimicrobial activity of both compounds was observed when P. gingivalis was grown in co-cultures with epithelial HeLa cells under low-iron/heme conditions, which mimic those occurring in vivo. 24DNTAM was more effective against P. gingivalis, but exhibited higher cytotoxic activity against epithelial and red blood cells, as compared with 24DNDMTAM. We conclude that both hemiaminals might originate a novel group of biologically important molecules.

Immune response of macrophages induced by Porphyromonas gingivalis requires HmuY protein.

The main etiologic agent and a key pathogen responsible for initiation and progression of chronic periodontitis is Porphyromonas gingivalis. We examined the role of P. gingivalis, with particular interest to HmuY protein, in expression of genes involved in Toll-like receptor (TLR)-induced signaling pathways using cell-based infection model. U937 and THP-1 cells differentiated toward macrophages by PMA treatment responded to P. gingivalis-caused infection in slightly different gene expression pattern, mainly by higher expression of genes encoding NF-κB, TLR7, TLR2, TLR8, pro-inflammatory cytokines (IL-1ß, IL-6, TNFα), anti-inflammatory cytokine (IL-10), and chemokines (CCL3L1, CCL4, CXCL10, CXCL11, PTX3). P. gingivalis lacking functional hmuY gene stimulates immune response of macrophages, albeit in a different manner as compared with the wild-type strain, mainly by lower expression of genes encoding NF-κB, IL-1ß, IL-10, CD80, PTX3, and CCL31L. The purified HmuY protein alone induced expression of genes encoding IL-6, IL-10, TNFα, CCL3L1, and CCL4. We conclude that macrophages respond to P. gingivalis infection mostly by TLR7-induced pathway(s). Moreover, P. gingivalis HmuY is one of important virulence factors, which allows P. gingivalis for in vivo growth in the heme-limited host environment, resulting in efficient immune response of macrophages.

HmuY is an important virulence factor for Porphyromonas gingivalis growth in the heme-limited host environment and infection of macrophages.

Porphyromonas gingivalis, the main etiologic agent and key pathogen responsible for initiation and progression of chronic periodontitis, is a haem auxotroph, and the uptake of this compound is essential for its survival and the ability to establish an infection. The aim of this study was to examine the role of a hemophore-like HmuY protein in P. gingivalis growth and infection of macrophages. Inactivation of the hmuY gene caused reduced P. gingivalis growth in vitro in the presence of serum as a heme sole source, as well as in vivo co-cultures with THP-1-derived macrophages. This resulted in diminished invasion efficiency of macrophages by live bacteria lacking functional hmuY gene. Both features were partially restored after addition of the purified HmuY protein, which was internalized when added either together with the hmuY mutant strain or alone to macrophage cultures. We conclude that HmuY is an important virulence factor of P. gingivalis for infection of macrophages in a heme-limited host environment.

UDP-galactose (SLC35A2) and UDP-N-acetylglucosamine (SLC35A3) Transporters Form Glycosylation-related Complexes with Mannoside Acetylglucosaminyltransferases (Mgats).

UDP-galactose transporter (UGT; SLC35A2) and UDP-N-acetylglucosamine transporter (NGT; SLC35A3) form heterologous complexes in the Golgi membrane. NGT occurs in close proximity to mannosyl (α-1,6-)-glycoprotein ß-1,6-N-acetylglucosaminyltransferase (Mgat5). In this study we analyzed whether NGT and both splice variants of UGT (UGT1 and UGT2) are able to interact with four different mannoside acetylglucosaminyltransferases (Mgat1, Mgat2, Mgat4B, and Mgat5). Using an in situ proximity ligation assay, we found that all examined glycosyltransferases are in the vicinity of these UDP-sugar transporters both at the endogenous level and upon overexpression. This observation was confirmed via the FLIM-FRET approach for both NGT and UGT1 complexes with Mgats. This study reports for the first time close proximity between endogenous nucleotide sugar transporters and glycosyltransferases. We also observed that among all analyzed Mgats, only Mgat4B occurs in close proximity to UGT2, whereas the other three Mgats are more distant from UGT2, and it was only possible to visualize their vicinity using proximity ligation assay. This strongly suggests that the distance between these protein pairs is longer than 10 nm but at the same time shorter than 40 nm. This study adds to the understanding of glycosylation, one of the most important post-translational modifications, which affects the majority of macromolecules. Our research shows that complex formation between nucleotide sugar transporters and glycosyltransferases might be a more common phenomenon than previously thought.

Porphyromonas gingivalis HmuY stimulates expression of Bcl-2 and Fas by human CD3+ T cells.

BACKGROUND: Apoptosis is a highly controlled process of cell death that can be induced by periodontopathogens. The present study aimed to investigate the expression of Fas and Bcl-2 proteins by CD3+ T cells in vitro under stimulation by total Porphyromonas gingivalis antigens and purified recombinant P. gingivalis HmuY protein. RESULTS: CD3+ T cells derived from CP patients and stimulated with HmuY expressed higher levels of Bcl-2 compared to identical cells stimulated with P. gingivalis crude extract or cells derived from NP control subjects (p = 0.043). CONCLUSION: The authors hypothesize that P. gingivalis HmuY plays a role in the pathogenesis of chronic periodontitis, possibly by reducing or delaying apoptosis in T cells through a pathway involving the Bcl-2 protein.

UDP-N-acetylglucosamine transporter (SLC35A3) regulates biosynthesis of highly branched N-glycans and keratan sulfate.

SLC35A3 is considered the main UDP-N-acetylglucosamine transporter (NGT) in mammals. Detailed analysis of NGT is restricted because mammalian mutant cells defective in this activity have not been isolated. Therefore, using the siRNA approach, we developed and characterized several NGT-deficient mammalian cell lines. CHO, CHO-Lec8, and HeLa cells deficient in NGT activity displayed a decrease in the amount of highly branched tri- and tetraantennary N-glycans, whereas monoantennary and diantennary ones remained unchanged or even were accumulated. Silencing the expression of NGT in Madin-Darby canine kidney II cells resulted in a dramatic decrease in the keratan sulfate content, whereas no changes in biosynthesis of heparan sulfate were observed. We also demonstrated for the first time close proximity between NGT and mannosyl (α-1,6-)-glycoprotein ß-1,6-N-acetylglucosaminyltransferase (Mgat5) in the Golgi membrane. We conclude that NGT may be important for the biosynthesis of highly branched, multiantennary complex N-glycans and keratan sulfate. We hypothesize that NGT may specifically supply ß-1,3-N-acetylglucosaminyl-transferase 7 (ß3GnT7), Mgat5, and possibly mannosyl (α-1,3-)-glycoprotein ß-1,4-N-acetylglucosaminyltransferase (Mgat4) with UDP-GlcNAc.

The Porphyromonas gingivalis HmuY haemophore binds gallium(iii), zinc(ii), cobalt(iii), manganese(iii), nickel(ii), and copper(ii) protoporphyrin IX but in a manner different to iron(iii) protoporphyrin IX.

Porphyromonas gingivalis, a major etiological agent of chronic periodontitis, acquires haem from host haemoproteins through a haem transporter HmuR and a haemophore HmuY. The aim of this study was to analyse the binding specificity of HmuY towards non-iron metalloporphyrins which may be employed as antimicrobials to treat periodontitis. HmuY binds gallium(iii), zinc(ii), cobalt(iii), manganese(iii), nickel(ii), and copper(ii) protoporphyrin IX but in a manner different to iron(iii) protoporphyrin IX which uses His(134) and His(166) as axial ligands. The metal ions in Ga(iii)PPIX and Zn(ii)PPIX can accept only His(166) as an axial ligand, whereas nickel(ii) and copper(ii) interact exclusively with His(134). Two forms of pentacoordinate manganese(iii) are present in the Mn(iii)PPIX-HmuY complex since the metal accepts either His(134) or His(166) as a single axial ligand. The cobalt ion is hexacoordinate in the Co(iii)PPIX-HmuY complex and binds His(134) and His(166) as axial ligands; however, some differences in their environments exist. Despite different coordination modes of the central metal ion, gallium(iii), zinc(ii), cobalt(iii), and manganese(iii) protoporphyrin IX bound to the HmuY haemophore cannot be displaced by excess haem. All of the metalloporphyrins examined bind to a P. gingivalis wild-type strain with higher ability compared to a mutant strain lacking a functional hmuY gene, thus corroborating binding of non-iron metalloporphyrins to purified HmuY protein. Our results further clarify the basis of metalloporphyrin acquisition by P. gingivalis and add to understanding of the interactions with porphyrin derivatives which exhibit antimicrobial activity against P. gingivalis.

Porphyromonas gingivalis HmuY-induced production of interleukin-6 and IL-6 polymorphism in chronic periodontitis.

BACKGROUND: In chronic periodontitis (CP), the gene polymorphism of interleukin-6 (IL-6) to 174C/G has been associated with the altered production of this cytokine. The aim of this pilot study is to compare the allelic and genotypic frequencies in patients with CP with control individuals without periodontitis (NP) and to measure the production of IL-6 by whole blood cells stimulated with Porphyromonas gingivalis HmuY protein. METHODS: DNA was isolated from peripheral blood cells of 49 patients with CP and 60 control individuals classified as NP, and genotyping was performed by polymerase chain reaction using sequence-specific primers. Whole blood cells from 29 patients with CP and 30 control individuals were stimulated for 48 hours with HmuY, and IL-6 levels were measured using enzyme-linked immunosorbent assay. RESULTS: The proportion of individuals carrying the G allele at position -174 of the IL-6 gene was higher in the group with CP (85.7%) than in the normal control group (73.3%; P <0.03). P. gingivalis HmuY-induced production of IL-6 was higher in the group with CP (P <0.05). CONCLUSIONS: Our findings suggest that P. gingivalis HmuY may be associated with increased IL-6 production during CP. Furthermore, patients with periodontitis and individuals with higher HmuY-induced production of IL-6 show a high frequency of the G allele at position -174.

Gallium(III), cobalt(III) and copper(II) protoporphyrin IX exhibit antimicrobial activity against Porphyromonas gingivalis by reducing planktonic and biofilm growth and invasion of host epithelial cells.

Porphyromonas gingivalis acquires heme for growth, and initiation and progression of periodontal diseases. One of its heme acquisition systems consists of the HmuR and HmuY proteins. This study analyzed the antimicrobial activity of non-iron metalloporphyrins against P. gingivalis during planktonic growth, biofilm formation, epithelial cell adhesion and invasion, and employed hmuY, hmuR and hmuY-hmuR mutants to assess the involvement of HmuY and HmuR proteins in the acquisition of metalloporphyrins. Iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) supported planktonic growth of P. gingivalis cells, biofilm accumulation, as well as survival, adhesion and invasion of HeLa cells in a way analogous to protoheme. In contrast, cobalt(III), gallium(III) and copper(II) protoporphyrin IX exhibited antimicrobial activity against P. gingivalis, and thus represent potentially useful antibacterial compounds with which to target P. gingivalis. P. gingivalis hmuY, hmuR and hmuY-hmuR mutants showed decreased growth and infection of epithelial cells in the presence of all metalloporphyrins examined. In conclusion, the HmuY protein may not be directly involved in transport of free metalloporphyrins into the bacterial cell, but it may also play a protective role against metalloporphyrin toxicity by binding an excess of these compounds.

Iron(III) mesoporphyrin IX and iron(III) deuteroporphyrin IX bind to the Porphyromonas gingivalis HmuY hemophore.

Porphyromonas gingivalis acquires heme through an outer-membrane heme transporter HmuR and heme-binding hemophore-like lipoprotein HmuY. Here, we compare binding of iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) to HmuY with that of iron(III) protoporphyrin IX (protoheme) and protoporphyrin IX (PPIX) using spectroscopic methods. In contrast to PPIX, mesoheme and deuteroheme enter the HmuY heme cavity and are coordinated by His134 and His166 residues in a fully analogous way to protoheme binding. However, in the case of deuteroheme two forms of HmuY-iron porphyrin complex were observed differing by a 180° rotation of porphyrin about the α-γ-meso-carbon axis. Since the use of porphyrins either as active photosensitizers or in combination with antibiotics may have therapeutic value for controlling bacterial growth in vivo, it is important to compare the binding of heme derivatives to HmuY.

HmuY haemophore and gingipain proteases constitute a unique syntrophic system of haem acquisition by Porphyromonas gingivalis.

Haem (iron protoporphyrin IX) is both an essential growth factor and virulence regulator for the periodontal pathogen Porphyromonas gingivalis, which acquires it mainly from haemoglobin via the sequential actions of the R- and K-specific gingipain proteases. The haem-binding lipoprotein haemophore HmuY and its cognate receptor HmuR of P. gingivalis, are responsible for capture and internalisation of haem. This study examined the role of the HmuY in acquisition of haem from haemoglobin and the cooperation between HmuY and gingipain proteases in this process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to wrest haem from immobilised methaemoglobin and deoxyhaemoglobin. Haem extraction from oxyhaemoglobin was facilitated after oxidation to methaemoglobin by pre-treatment with the P. gingivalis R-gingipain A (HRgpA). HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp). This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin. In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.

Diphosphonucleotide phosphatase/phosphodiesterase (PPD1) from yellow lupin (Lupinus luteus L.) contains an iron-manganese center.

Yellow lupin diphosphonucleotide phosphatase/phosphodiesterase (PPD1) represents a novel group of enzymes. Here we report that it possesses one iron atom and one manganese atom (1:1 molar ratio) per subunit. The enzyme exhibits visible absorption maximum at approximately 530 nm. Prolonged oxidation of PPD1 leads to loss of the charge-transfer band and catalytic activity, whereas after reduction PPD1 remains active. Replacement of conserved amino-acid residues coordinating metals results in the loss of enzymatic activity. Despite low amino-acid sequence homology of PPD1 to well-characterized approximately 55-kDa purple acid phosphatases, their overall fold, topology of active center and metal content are highly similar.

Heme environment in HmuY, the heme-binding protein of Porphyromonas gingivalis.

Porphyromonas gingivalis, a Gram-negative anaerobic bacterium implicated in the development and progression of chronic periodontitis, acquires heme for growth by a novel mechanism composed of HmuY and HmuR proteins. The aim of this study was to characterize the nature of heme binding to HmuY. The protein was expressed, purified and detailed investigations using UV-vis absorption, CD, MCD, and (1)H NMR spectroscopy were carried out. Ferric heme bound to HmuY may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. Heme complexed to HmuY, with a midpoint potential of 136mV, is in a low-spin Fe(III) hexa-coordinate environment. Analysis of heme binding to several single and double HmuY mutants with the methionine, histidine, cysteine, or tyrosine residues replaced by an alanine residue identified histidines 134 and 166 as potential heme ligands.

N-glycosylation sites of plant purple acid phosphatases important for protein expression and secretion in insect cells.

Analysis of plant purple acid phosphatases (PAPs) showed high conservation and different distribution of N-glycosylation sites. Oligosaccharide structures of Lupinus luteus acid phosphatase (Lu_AP) produced in insect cells were determined. Mutant Lu_AP and Phaseolus vulgaris (Ph_AP) phosphatases lacking possibility of N-glycosylation at highly conserved sites were generated and expressed in insect cells. A role for N-glycosylation in the stability of PAPs was indicated by unsuccessful attempts to secrete Ph_AP and Lu_AP mutants generated by replacing Asn residues of conserved glycosylation sequons by Ser residues either singly or in combination. We showed that Ph_AP belongs to the group of glycoproteins that require occupancy of all highly conserved glycosylation sites for secretion, whereas replacing of the third position of the glycosylation sequon indicated that Lu_AP may tolerate the absence of some N-glycans. However, the N-glycan located at the polypeptide C-terminus was crucial for secretion of both enzymes. PAP specific activity of glycosylation mutants successfully secreted was similar to the wild-type recombinant proteins.