Sialic acid metabolism in sialuria fibroblasts

Sialuria is a rare inborn error of metabolism caused by excessive synthesis of sialic acid (N-acetylneuraminic acid, NeuAc). Fibroblasts cultured from the three known cases of sialuria contained 70-200-fold increases in soluble sialic acid, but normal concentrations of bound sialic acid. The sialic acid appeared in the cytosolic fraction of the cells on differential centrifugation, and was susceptible to borohydride reduction, suggesting that accumulated sialic acid was in the form of NeuAc and not CMP-NeuAc. In biochemical studies, CMP-NeuAc (50 microM) inhibited the UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase of normal fibroblasts by 84-100%, but inhibited the epimerase from sialuria cells by only 19-31%. Feeding sialuria cells up to 5 mM D-glucosamine for 72 h increased free sialic acid content 20-60%, but normal cells were unaffected by this treatment. Cytidine feeding (5 mM, 72 h) reduced the NeuAc content of sialuria cells, initially 112, 104, and 266 nmol/mg protein, by 63-71 nmol/mg protein; CMP-NeuAc concentrations, initially 4, 2, and 5 nmol/mg protein, increased by 14-33 nmol/mg protein. Consequently, the total cellular content of soluble sialic acid (NeuAc + CMP-NeuAc) was lowered 14-46% by cytidine feeding. The inheritance pattern of sialuria has not been determined. However, cells from both parents of one sialuria patient contained normal concentrations of free sialic acid, and the parental epimerase activity also responded normally to CMP-NeuAc. We conclude that the basic biochemical defect in all known cases of sialuria is a failure of CMP-NeuAc to feedback-inhibit UDP-GlcNAc 2-epimerase and cytidine feeding can lower the intracellular soluble sialic acid concentration of sialuria cells.     

Binding of zona binding inhibitory factor-1 (ZIF-1) from human follicular fluid on spermatozoa

Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.     

Structure-function analysis of PrsA reveals roles for the parvulin-like and flanking N- and C-terminal domains in protein folding and secretion in Bacillus subtilis

The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.     

1H, 13C and 15N resonance assignments of the major extracytoplasmic domain of the cell shape-determining protein MreC from Bacillus subtilis

MreB, MreC and MreD are essential cell shape-determining morphogenetic proteins in Gram-positive and in Gram-negative bacteria. While MreB, the bacterial homologue of the eukaryotic cytoskeletal protein actin, has been extensively studied, the roles of MreC and MreD are less well understood. They both are transmembrane proteins. MreC has a predicted single transmembrane domain and the C-terminal part outside the cell membrane. MreC probably functions as a link between the intracellular cytoskeleton and the cell wall synthesizing machinery which is located at the outer surface of the cell membrane. Also proteins involved in cell wall synthesis participate in cell morphogenesis. How these two processes are coordinated is, however, poorly understood. Bacillus subtilis (BS), a non-pathogenic Gram-positive bacterium, is widely used as a model for Gram-positive pathogens, e.g. Staphylococcus aureus (SA). Currently, the structures of MreC from BS and SA are not known. As part of our efforts to elucidate the structure–function relationships of the morphogenetic protein complexes in Gram-positive bacteria, we present the backbone and side chain resonance assignments of the extracytoplasmic domain of MreC from BS.     

Differences in glycosylation and sperm-egg binding inhibition of pregnancy-related glycodelin

Glycodelin is a glycoprotein produced in many glands, particularly those of reproductive tissues. It appears as different glycoforms in amniotic fluid (glycodelin-A) and seminal plasma (glycodelin-S), but only glycodelin-A inhibits gamete adhesion. In the present study, glycodelin from secretory-phase endometrium, first-trimester pregnancy decidua, and midtrimester amniotic fluid was studied with respect to physicochemical properties, including glycosylation patterns and inhibitory activity of sperm-egg binding. Purified glycodelins from all these sources were similar in isoelectric focusing and in lectin immunoassays using lectins from Wisteria floribunda and Sambucus nigra. Likewise, the glycodelins inhibited sperm-egg binding in a dose-dependent manner, as measured by hemizona-binding assay. However, subtle quantitative physicochemical and biological differences were found between glycodelins from different sources as well as within the same tissue/fluid between different individuals. Differences were most pronounced between endometrial glycodelins from nonpregnancy and first-trimester pregnancy. The glycan structures studied by fast-atom bombardment mass spectrometry of individual amniotic fluid glycodelin-A samples also showed interindividual quantitative differences. In conclusion, glycodelins from different female reproductive tract tissues and amniotic fluid share substantial similarity, allowing all of them to be called glycodelin-A. However, these glycodelins exhibit quantitative physicochemical and functional differences between different sources and individuals.     

Defective lysosomal egress of free sialic acid (N-acetylneuraminic acid) in fibroblasts of patients with infantile free sialic acid storage disease

Egress of free NeuAc from normal lysosome-rich granular fractions was assessed at NeuAc concentrations of up to 221 pmol/hexosaminidase unit, achieved by exposure of growing fibroblasts to 40-125 nM N-acetylmannosamine for up to 7 days. The normal velocity of NeuAc egress increased with NeuAc loading and with temperature, exhibiting a Q10 of 2.4, characteristic of carrier-mediated transport. Fibroblasts cultured from five patients with infantile free sialic acid storage disease (ISSD) contained approximately 139 nmol of free NeuAc/mg of whole cell protein, or 100 times the normal level. Differential centrifugation, as well as density gradient analysis using 25% Percoll, showed that the stored NeuAc cosedimented with the lysosomal enzyme beta-hexosaminidase. The velocity of appearance of free NeuAc outside ISSD granular fractions was negligible, even at initial loading levels of up to 3500 pmol/hexosaminidase unit. The lack of egress from ISSD granular fractions was found for both endogenous and N-acetylmannosamine-derived NeuAc. Fibroblasts from ISSD parents did not accumulate excess free NeuAc and did not display a velocity of NeuAc egress significantly different from normal. The defect in ISSD, like that in Salla disease, appears to be an impairment of carrier-mediated transport of free NeuAc across the lysosomal membrane. Clinical and biochemical differences between Salla disease and ISSD may reflect differences in the amount of residual NeuAc transport capacity.