SLC35A2 Deficiency Promotes an Epithelial-to-Mesenchymal Transition-like Phenotype in Madin-Darby Canine Kidney Cells.

In mammalian cells, SLC35A2 delivers UDP-galactose for galactosylation reactions that take place predominantly in the Golgi lumen. Mutations in the corresponding gene cause a subtype of a congenital disorder of glycosylation (SLC35A2-CDG). Although more and more patients are diagnosed with SLC35A2-CDG, the link between defective galactosylation and disease symptoms is not fully understood. According to a number of reports, impaired glycosylation may trigger the process of epithelial-to-mesenchymal transition (EMT). We therefore examined whether the loss of SLC35A2 activity would promote EMT in a non-malignant epithelial cell line. For this purpose, we knocked out the SLC35A2 gene in Madin-Darby canine kidney (MDCK) cells. The resulting clones adopted an elongated, spindle-shaped morphology and showed impaired cell-cell adhesion. Using qPCR and western blotting, we revealed down-regulation of E-cadherin in the knockouts, while the fibronectin and vimentin levels were elevated. Moreover, the knockout cells displayed reorganization of vimentin intermediate filaments and altered subcellular distribution of a vimentin-binding protein, formiminotransferase cyclodeaminase (FTCD). Furthermore, depletion of SLC35A2 triggered Golgi compaction. Finally, the SLC35A2 knockouts displayed increased motility and invasiveness. In conclusion, SLC35A2-deficient MDCK cells showed several hallmarks of EMT. Our findings point to a novel role for SLC35A2 as a gatekeeper of the epithelial phenotype.

The Solute Carrier MFSD1 Decreases the Activation Status of ß1 Integrin and Thus Tumor Metastasis.

Solute carriers are increasingly recognized as participating in a plethora of pathologies, including cancer. We describe here the involvement of the orphan solute carrier Major Facilitator Superfamily Domain-containing protein 1 (MFSD1) in the regulation of tumor cell migration. Loss of MFSD1 enabled higher levels of metastasis in experimental and spontaneous metastasis mouse models. We identified an increased migratory potential in MFSD1-/- tumor cells which was mediated by increased focal adhesion turnover, reduced stability of mature inactive ß1 integrin, and the resulting increased integrin activation index. We show that MFSD1 promoted recycling to the cell surface of endocytosed inactive ß1 integrin and thereby protected ß1 integrin from proteolytic degradation; this led to dampening of the integrin activation index. Furthermore, downregulation of MFSD1 expression was observed during the early steps of tumorigenesis, and higher MFSD1 expression levels correlate with a better cancer patient prognosis. In sum, we describe a requirement for endolysosomal MFSD1 in efficient ß1 integrin recycling to suppress tumor cell dissemination.

Modified secreted alkaline phosphatase as an improved reporter protein for N-glycosylation analysis.

N-glycosylation is a common posttranslational modification of proteins in eukaryotic cells. The modification is often analyzed in cells which are able to produce extracellular, glycosylated proteins. Here we report an improved method of the use of genetically modified, secreted alkaline phosphatase (SEAP) as a reporter glycoprotein which may be used for glycoanalysis. Additional N-glycosylation sites introduced by site-directed mutagenesis significantly increased secretion of the protein. An improved purification protocol of recombinant SEAP from serum or serum-free media is also proposed. The method enables fast and efficient separation of reporter glycoprotein from a relatively small amount of medium (0.5-10 ml) with a high recovery level. As a result, purified SEAP was ready for enzymatic de-glycosylation without buffer exchange, sample volume reductions or other procedures, which are usually time-consuming and may cause partial loss of the reporter glycoprotein.

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 Insights into Selected Disease-Causing Mutations within the SLC35A1 Gene Encoding the CMP-Sialic Acid Transporter.

Congenital disorders of glycosylation (CDG) are a group of rare genetic and metabolic diseases caused by alterations in glycosylation pathways. Five patients bearing CDG-causing mutations in the SLC35A1 gene encoding the CMP-sialic acid transporter (CST) have been reported to date. In this study we examined how specific mutations in the SLC35A1 gene affect the protein's properties in two previously described SLC35A1-CDG cases: one caused by a substitution (Q101H) and another involving a compound heterozygous mutation (T156R/E196K). The effects of single mutations and the combination of T156R and E196K mutations on the CST's functionality was examined separately in CST-deficient HEK293T cells. As shown by microscopic studies, none of the CDG-causing mutations affected the protein's proper localization in the Golgi apparatus. Cellular glycophenotypes were characterized using lectins, structural assignment of N- and O-glycans and analysis of glycolipids. Single Q101H, T156R and E196K mutants were able to partially restore sialylation in CST-deficient cells, and the deleterious effect of a single T156R or E196K mutation on the CST functionality was strongly enhanced upon their combination. We also revealed differences in the ability of CST variants to form dimers. The results of this study improve our understanding of the molecular background of SLC35A1-CDG cases.

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.

SLC35A2-CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals.

Pathogenic de novo variants in the X-linked gene SLC35A2 encoding the major Golgi-localized UDP-galactose transporter required for proper protein and lipid glycosylation cause a rare type of congenital disorder of glycosylation known as SLC35A2-congenital disorders of glycosylation (CDG; formerly CDG-IIm). To date, 29 unique de novo variants from 32 unrelated individuals have been described in the literature. The majority of affected individuals are primarily characterized by varying degrees of neurological impairments with or without skeletal abnormalities. Surprisingly, most affected individuals do not show abnormalities in serum transferrin N-glycosylation, a common biomarker for most types of CDG. Here we present data characterizing 30 individuals and add 26 new variants, the single largest study involving SLC35A2-CDG. The great majority of these individuals had normal transferrin glycosylation. In addition, expanding the molecular and clinical spectrum of this rare disorder, we developed a robust and reliable biochemical assay to assess SLC35A2-dependent UDP-galactose transport activity in primary fibroblasts. Finally, we show that transport activity is directly correlated to the ratio of wild-type to mutant alleles in fibroblasts from affected individuals.

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.

An insight into the orphan nucleotide sugar transporter SLC35A4.

SLC35A4 has been classified in the SLC35A subfamily based on amino acid sequence homology. Most of the proteins belonging to the SLC35 family act as transporters of nucleotide sugars. In this study, the subcellular localization of endogenous SLC35A4 was determined via immunofluorescence staining, and it was demonstrated that SLC35A4 localizes mainly to the Golgi apparatus. In silico topology prediction suggests that SLC35A4 has an uneven number of transmembrane domains and its N-terminus is directed towards the Golgi lumen. However, an experimental assay refuted this prediction: SLC35A4 has an even number of transmembrane regions with both termini facing the cytosol. In vivo interaction analysis using the FLIM-FRET approach revealed that SLC35A4 neither forms homomers nor associates with other members of the SLC35A subfamily except SLC35A5. Additional assays demonstrated that endogenous SLC35A4 is 10 to 40nm proximal to SLC35A2 and SLC35A3. To determine SLC35A4 function SLC35A4 knock-out cells were generated with the CRISPR-Cas9 approach. Although no significant changes in glycosylation were observed, the introduced mutation influenced the subcellular distribution of the SLC35A2/SLC35A3 complexes. Additional FLIM-FRET experiments revealed that overexpression of SLC35A4-BFP together with SLC35A3 and the SLC35A2-Golgi splice variant negatively affects the interaction between the two latter proteins. The results presented here strongly indicate a modulatory role for SLC35A4 in intracellular trafficking of SLC35A2/SLC35A3 complexes.

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.

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.

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.

Isolation, characterization and cDNA sequencing of acrosin from turkey spermatozoa.

Acrosin (EC 3.4.21.10) is serine proteinase localized in the sperm acrosome and considered to play an essential role in fertilization. In contrast to mammalian, there are only limited data concerning avian acrosin, mostly focused on the characterization of mature enzyme. In the present study, acrosin was isolated from turkey spermatozoa using gel filtration in the presence of 4 M urea at acidic pH. N-terminal Edman sequencing allowed the identification of the first 26 N-terminal amino acids: VVGGTEALHG SWPWIVSIQNPRFAGT. This sequence was used to construct primers and obtain a cDNA sequence from the testis. The amino acid sequence deduced from the cDNA shows that turkey acrosin is initially synthesized as prepro-protein with 19-residue signal peptide. This signal sequence is followed by a 327-residue sequence corresponding to the acrosin zymogen. Turkey proacrosin does not contain a proline-rich segment at the C-terminal portion. Mature turkey acrosin is a two-chain molecule consisting of light and heavy chains and was found to be glycoprotein. The proacrosin/acrosin system exists in turkey spermatozoa and this system can be activated similarly to that of mammals. The high value of association constant strongly suggests that acrosin activity in turkeys can be controlled by a seminal plasma Kazal inhibitor under physiological conditions.

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.