Precocious loss of cortical granules during mouse oocyte meiotic maturation and correlation with an egg-induced modification of the zona pellucida

Fertilization results in cortical granule exocytosis, which is thought to be involved in modifications of the zona pellucida that constitute the zona pellucida block to polyspermy. A previous report demonstrated that a decrease in the number of Lens culinaris agglutinin-staining granules, which are likely to be cortical granules, occurred during in vivo mouse oocyte maturation with arrest at metaphase II, as well as the formation of a cortical granule-free domain in the area of the metaphase II spindle (T. Ducibella, E. Anderson, D.F. Albertini, J. Aalberg, and S. Rangarajan, 1988, Dev. Biol. 130, 184-197). We extend these observations by reporting here that germinal vesicle-intact oocytes matured in vitro to metaphase II in either the absence or the presence of serum develop a cortical granule-free domain and have reduced numbers of cortical granules when compared to germinal vesicle-intact oocytes; these changes are similar to those of oocytes matured in vivo. The reduction in the number of cortical granules requires germinal vesicle breakdown, since it is prevented by dibutyryl cAMP, which inhibits germinal vesicle breakdown in vitro. The ability of oocytes to respond to the calcium ionophore A23187 with a reduction in the number of cortical granules is also associated with meiotic maturation and develops between 7 and 12 hr after initiation of maturation. The maturation-associated reduction in the number of cortical granules is likely to represent cortical granule exocytosis, since this reduction is accompanied by the formation of a cortical granule-free domain and a conversion of ZP2 to ZP2f when the oocytes are matured in vitro in serum-free medium; this zona pellucida modification occurs following fertilization and is thought to be due to cortical granule exocytosis. In contrast, the loss of cortical granules and development of the cortical granule-free domain of oocytes matured in vitro in the presence of serum is not accompanied by the modification of ZP2. The inhibitory effect of serum on the ZP2 modification may afford in vivo a physiological mechanism to prevent a precocious modification of the zona pellucida that could result in a premature block to polyspermy and hence inhibit fertilization.     

Role of water molecules and ion pairs in Dps and related ferritin-like structures

A comparative study of water molecules and ion pairs in 11 Dps protein structures has been carried out. The invariant and common water molecules, the conserved residues interacting with them and the conserved ion pairs have been analyzed. Certain water molecules found on the interfaces between subunits are highly conserved and may be implicated in flexibility or continuing association of the subunits of the structure. It is possible that the water molecules, ion pairs and the special case of a water mediated charged network through a single water molecule are involved in maintaining the stability of the protein.     

Structural Analysis of the Core Region of O-Lipopolysaccharide of Porphyromonas gingivalis from Mutants Defective in O-Antigen Ligase and O-Antigen Polymerase

Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPSs), O-LPS and A-LPS. Here, we elucidate the structure of the core oligosaccharide (OS) of O-LPS from two mutants of P. gingivalis W50, ΔPG1051 (WaaL, O-antigen ligase) and ΔPG1142 (O-antigen polymerase), which synthesize R-type LPS (core devoid of O antigen) and SR-type LPS (core plus one repeating unit of O antigen), respectively. Structural analyses were performed using one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy in combination with composition and methylation analysis. The outer core OS of O-LPS occurs in two glycoforms: an “uncapped core,” which is devoid of O polysaccharide (O-PS), and a “capped core,” which contains the site of O-PS attachment. The inner core region lacks l(d)-glycero-d(l)-manno-heptosyl residues and is linked to the outer core via 3-deoxy-d-manno-octulosonic acid, which is attached to a glycerol residue in the outer core via a monophosphodiester bridge. The outer region of the “uncapped core” is attached to the glycerol and is composed of a linear α-(1→3)-linked d-Man OS containing four or five mannopyranosyl residues, one-half of which are modified by phosphoethanolamine at position 6. An amino sugar, α-d-allosamine, is attached to the glycerol at position 3. In the “capped core,” there is a three- to five-residue extension of α-(1→3)-linked Man residues glycosylating the outer core at the nonreducing terminal residue. β-d-GalNAc from the O-PS repeating unit is attached to the nonreducing terminal Man at position 3. The core OS of P. gingivalis O-LPS is therefore a highly unusual structure, and it is the basis for further investigation of the mechanism of assembly of the outer membrane of this important periodontal bacterium.Porphyromonas gingivalis is a gram-negative anaerobe which is strongly implicated in the etiology of periodontal disease. Several putative virulence factors are produced by this organism. These virulence factors include the cysteine proteases Arg-gingipains (Rgps) and Lys-gingipain (Kgp) specific for Arg-X and Lys-X peptide bonds, respectively, which are capable of degrading several host proteins (56), and lipopolysaccharide (LPS), which has the potential to cause an inflammatory response in the periodontal tissues of the host. These factors are important antigens in patients with periodontal disease and may account for a considerable proportion of the immune response directed against P. gingivalis (58).LPS is a major constituent of the outer membrane of gram-negative bacteria and facilitates interactions with the external environment. It consists of three regions: a hydrophobic lipid A embedded in the outer leaflet of the outer membrane, a core oligosaccharide (OS), and the O-polysaccharide (O-PS) side chain composed of several repeating units. The hydrophobic lipid A serves as an anchor for the LPS and consists of β-1,6-linked d-glucosamine disaccharide, which is usually phosphorylated at the 1 and/or 4′ positions and N and/or O acylated at positions 2, 3, 2′, and 3′ with various amounts of fatty acids. The rest of the LPS molecule projects from the surface. The core region is attached to lipid A and is composed of ∼10 sugars in most bacteria studied to date and can be further subdivided into an inner core and an outer core. The inner core usually contains l(d)-glycero-d-(l)-manno-heptose and 3-deoxy-d-manno-octulosonic acid (Kdo) residues, whereas the outer core is usually composed of hexoses. Attached to the outer core are the repeating units of O antigen (O-PS), which vary in composition, stereochemistry, and the sequence of O-glycosidic linkages between bacterial strains and thereby give rise to O-serotype specificity within bacterial species. Attachment of O antigen to core lipid A results in “smooth” LPS (S-type LPS), whereas LPS lacking O antigen is “rough” LPS (R-type LPS). Attachment of one repeating unit of O-PS to core lipid A results in SR-LPS (core-plus-one repeating unit) (41, 47, 48). In addition, the outer core OS region can be either “uncapped” or “capped.” The “uncapped” core OS is devoid of O-PS repeating units, whereas the “capped” core OS contains attached O-PS repeating units (47, 53) due to modifications in the outer core region.P. gingivalis W50 was originally thought to synthesize a single LPS composed of a tetrasaccharide repeating unit in the O-PS, [→6)-α-d-Glcp-(1→4)-α-l-Rhap-(1→3)-β-d-GalNAc-(1→3)-α-d-Galp-(1→], which is modified by phosphoethanolamine (PEA) at position 2 of Rha in a nonstoichiometric manner (43). However, a second LPS in this organism, namely A-LPS (49), which has a phosphorylated mannan-containing anionic polysaccharide (A-PS), was identified in our laboratory. The A-PS repeating unit is built up of a phosphorylated branched d-Man-containing oligomer composed of an α1→6-linked d-mannose backbone to which α1→2-linked d-Man side chains of different lengths (one or two residues) are attached at position 2. One of the side chains contains Manα1→2-Manα-1-phosphate linked via phosphorus to a backbone Man residue at position O-2. Although A-LPS is predominantly composed of α-d-mannose residues, it cannot be referred to as a homopolymer due to the presence of Manα1→2Manα1-phosphate-containing OS side chains forming a nonglycosidic linkage between the backbone α-mannose and side chains. Hence, it is likely that the synthesis of A-PS (A-LPS) occurs via a “wzy-dependent” pathway in which repeating units formed on the cytoplasmic face of the inner membrane are polymerized at the periplasmic face following transport or flipping across the cytoplasmic membrane. A-LPS cross-reacts with monoclonal antibody (MAb) 1B5 raised against one of the isoforms of Arg-gingipains, a family of differentially glycosylated cysteine proteases (14, 19). Deglycosylation of the cross-reacting Rgps with anhydrous trifluoromethane sulfonic acid abolishes their immunoreactivity to MAb 1B5, indicating that this antibody recognizes a carbohydrate-containing epitope also present in A-LPS (14, 44). Hence, there appear to be common elements in the biosynthesis of A-LPS and the Arg-gingipains of this organism.Inactivation of P. gingivalis waaL (PG1051, O-antigen ligase) abolishes the synthesis of both O-LPS and A-LPS (49). Hence, the WaaL O-antigen ligase appears to have dual specificity and is capable of ligating both O-PS and A-PS chains to core lipid A. The dual specificity of WaaL in the final step of LPS biosynthesis has also been demonstrated in the synthesis of Escherichia coli O-LPS and M_LPS (38) and for Pseudomonas aeruginosa A-band and B-band LPSs (1).However, the linkage between O-PS and A-PS and core OS has not been identified in P. gingivalis. In this paper, we describe a structural investigation of the core OS of O-LPS in which we used R-LPS prepared from ΔPG1051 (49) and ΔPG1142 (putative O-antigen polymerase), which we hypothesized would synthesize an SR-LPS (core plus one repeating unit) (60). The putative O-antigen polymerase encoded at PG1142 (42) is a phenylalanine-rich membrane protein consisting of 347 amino acids which shows 46% similarity over 297 amino acids to EpsK of Lactobacillus delbrueckii subsp. bulgaricus. EpsK is proposed to be a polymerase on the basis of homology and topological similarity to the O-antigen polymerase (Wzy) of E. coli and is required for the synthesis of an exopolysaccharide composed of Gal, Glc, and Rha (5:1:1) containing repeating units in L. delbrueckii (32). Application of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy and methylation and monosaccharide analyses using gas chromatography-mass spectrometry (GC-MS) to purified core-containing OSs isolated from LPS from ΔPG1051 and ΔPG1142 mutants enabled us to solve the LPS core structure of an oral gram-negative bacterium for the first time.     

Structural analysis of the polysaccharide from the lipopolysaccharide of Porphyromonas gingivalis strain W50.

The lipopolysaccharide (LPS) of Porphyromonas gingivalis is an important pro-inflammatory molecule in periodontal disease and a significant target of the host's specific immune response. In addition, we recently demonstrated using monoclonal antibodies that the Arg-gingipains of P. gingivalis are post-translationally modified with glycan chains that are immunologically related to an LPS preparation from this organism. In the present investigation, we determined the structure of the O-polysaccharide of P. gingivalis W50 that was fully characterized on the basis of 1D and 2D NMR (DQF-COSY, TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-31P HXTOCSY) and GC-MS data. These data allowed us to conclude that the O-polysaccharide is built up of the tetrasaccharide repeating sequence: -->6)-alpha-D-Glcp-(1-->4)-alpha-L-Rhap-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Galp-(1--> and carries a monophosphoethanolamine residue at position C-2 of the alpha-rhamnose residue in a nonstoichiometric (approximately 60%) amount. These data indicate that the O-polysaccharide of P. gingivalis LPS is composed of an unusually modified tetrasaccharide repeating unit.     

The development of mouse oocyte cortical reaction competence is accompanied by major changes in cortical vesicles and not cortical granule depth

The basis for the incompetence of the cortical reaction in germinal vesicle stage (GV) mouse oocytes was studied by evaluating cortical granules (CGs) and vesicles in GV and mature oocyte cortices. Dark and light CGs had a similar mean distance of 0.4-0.6 micron from the plasma membrane for GV and mature cortices. The cortex of mature oocytes had a large population of membrane-bounded, 0.1-1.0 micron (diameter) vesicles. More than three times as many vesicles were observed in the CG domains of mature oocytes as were observed in GV oocytes. This lack of cortical vesicles (with their potential to store calcium) and not CG depth may account for cortical reaction incompetence in GV oocytes.