Identification and characterization of podocalyxin--the major sialoprotein of the renal glomerular epithelial cell.

The glomerular epithelial polyanion is a specialized cell surface component found on renal glomerular epithelial cells (podocytes) that is rich in sialoprotein(s), as detected by staining with cationic dyes (colloidal iron, alcian blue) and wheat germ agglutinin (WGA). We have isolated rat glomeruli and analyzed their protein composition by SDS PAGE in 5-10% gradient gels. When the gels were stained with alcian blue or "Stains All," a single band with an apparent Mr of 140,000 was detected that also stained very prominently with silver, but not with Coomassie Blue. This band predominated in fluorograms of gels of isolated glomeruli that had been labeled in their sialic acid residues by periodate-[3H]borohydride. In lectin overlays, the 140-kilodalton (kd) band was virtually the only one that bound [125I]wheat germ agglutinin, and this binding could be prevented by predigestion with neuraminidase. [125I]Peanut lectin bound exclusively to the 140-kd band after neuraminidase treatment. An antibody was prepared that specifically recognizes only the 140-kd band by immunoprecipitation and immuneoverlay. By immunoperoxidase and immunogold techniques, it was localized to the surface coat of the glomerular epithelium and, less extensively, to that of endothelial cells. When analyzed (after electroelution from preparative SDS gels), the 140-kd band was found to contain approximately 20% hexose and approximately 4.5% sialic acid. These findings indicate that the 140-kd protein is the major sialoprotein of the glomerulus, and it is the only component of glomerular lysates with an affinity for cationic dyes and lectins identical to that defined histochemically for the epithelial polyanion in situ. Since this molecule is a major component of the cell coat or glycocalyx of the podocytes, we have called it "podocalyxin."

Topographical characterization of the DNA polymerase from Thermus aquaticus. Defining groups of inhibitor mAbs by epitope mapping and functional analysis using surface plasmon resonance.

Among 24 unique monoclonal antibodies (mAb) generated against Taq polymerase (TaqPol) 13 are potent inhibitors of polymerase activity. These antibodies have been sorted into groups defined by their topographical or functional properties using surface plasmon resonance-based methods to examine three different types of interactions. An epitope map of all the pairwise interactions among all 24 antiTaqPol antibodies revealed the surface of TaqPol as a complex space populated by isolated antigenic domains with no evident relationship to each other. 11 discrete epitopes or epitope clusters were defined and potent inhibitors bound to sites within seven of them. The second method examined the ability of antiTaqPol mAbs to bind to recombinant forms of the constituent functional domains of TaqPol, the N-terminal 5'-nuclease domain and the C-terminal polymerase domain. Most of the antibodies demonstrated a clear specificity for one domain or the other. The third method measured the ability of each mAb to block the interaction of TaqPol with a preformed, immobilized primer:template complex (PTC). Some antibodies had no effect on this interaction while others effectively blocked it. Together these latter two methods resolved many of the antibodies into five distinct groups. In addition, antibodies that bound to overlapping epitopes in the pairwise interaction analysis were members of the same group by their interaction with the polymerase fragment and PTC. Three groups of polymerase inhibitors were clearly resolved by these analyses: (1) those that recognize an epitope on the 5'-nuclease domain and have no effect on the interaction of TaqPol with PTC; (2) those that recognize an epitope on the polymerase domain and block the interaction of TaqPol and PTC; and (3) those that recognize an epitope on the polymerase domain, but have no effect on the interaction of TaqPol with PTC. The surface of TaqPol bears at least three antigenic regions that are topographically and functionally distinct and may correspond to sites for inhibition of different steps in the enzymatic activity of TaqPol.

Monoclonal antibodies prepared against the DNA polymerase from Thermus aquaticus are potent inhibitors of enzyme activity.

Recent interest in the unique properties of the DNA polymerase from Thermus aquaticus (TaqPol) has stemmed from its use in many laboratories for the polymerase chain reaction. We have produced a panel of nine distinct monoclonal antibodies to a recombinant form of TaqPol that have the following properties: (1) each binds TaqPol with high affinity (Kd < 10 nM); (2) eight of the nine arbitrarily selected monoclonal antibodies inhibit TaqPol activity completely; (3) the weak inhibitor is specific for TaqPol only while all eight strong inhibitors cross-react with the DNA polymerase from at least one other Thermus species as detected by either competitive ELISA, Western blotting, inhibition of enzyme activity or determination of binding by surface plasmon resonance; (4) these antibodies can be distinguished from each other by heavy chain class, cross-reactivity patterns, isoelectric points, and epitope mapping; and (5) these antibodies define seven non-overlapping epitopes. In addition, we show data from a preliminary experiment that demonstrates that at least one of these antibodies inhibits TaqPol by preventing DNA binding.

Development of a procedure for autoradiographic localization of carbonic anhydrase at the electron microscope level.

A method for preparing tissue suitable for electron microscope autoradiographic localization of carbonic anhydrase is described. Radioactivity is in the form of 3H-acetazolamide, a specific inhibitor of carbonic anhydrase. Tissues fixed in glutaraldehyde, dehydrated in ethylene glycol followed by cellosolve as a transition fluid, and embedded in epoxy resin, were found to retain most (74%) of the label. Electron micrographs of avian gastric mucosa prepared in this manner are shown. Other methods of preparation were explored and resulted in considerable losses of label or in inadequately preserved tissue. Light microscope autoradiographic localization of gastric mucosa, shell gland, chorioallantoic membrane, and skeletal muscle compare well with previous localizations.

Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction.

We demonstrate the utility of antibodies to the DNA polymerase from Thermus aquaticus (TaqPol) as thermolabile inhibitors of TaqPol activity. One of the limitations of the polymerase chain reaction (PCR) is the co-amplification of non-specific products caused by TaqPol activity on low stringency templates present in the initial cycle of PCR. We have used anti-TaqPol antibodies as thermolabile switches that inhibit TaqPol activity at low temperatures (20-40 degrees C) and release fully active TaqPol when they are inactivated by elevated temperatures in the PCR thermal cycling (70-98 degrees C). Several in a set of high affinity anti-TaqPol monoclonal antibodies fully inhibited TaqPol activity at 37 degrees C. The capacity for inhibition was ablated by incubation at temperatures high enough to denature antibodies but not sufficiently high to significantly reduce TaqPol activity. In a PCR model system, preincubation of TaqPol with these antibodies yielded PCR product consisting entirely of the intended product and the absence or significant reduction of non-specific products and primer dimers. In evaluation of clinical samples such antibody triggering yielded defined PCR product and higher sensitivity because of the absence of non-specific products.

Developmental regulation of processing alpha-mannosidases and "intersecting" N-acetylglucosaminyltransferase in Dictyostelium discoideum.

We have identified three developmentally regulated oligosaccharide-processing enzyme activities in Dictyostelium discoideum. Two different alpha-mannosidase activities present at extremely low levels in vegetative cells are expressed during development. The first of these activities (MI) rises sharply from 6 to 12 h of development whereas the second activity (MII) rises sharply from 12 to 18 h of development. MI acts on Man9GlcNAc, which it can degrade to Man5GlcNAc but is inactive toward p-nitrophenyl-alpha-D-mannoside (pnpMan). MII acts on pnpMan but not Man9GlcNAc. These activities are distinct from each other and from lysosomal alpha-mannosidase activity as demonstrated by pH optima, substrate specificity, sensitivity to inhibitors and divalent cations, developmental profiles, and solubility. The characteristics of these developmentally regulated alpha-mannosidase activities are similar to those of Golgi alpha-mannosidases I and II from higher eucaryotes, and they appear to catalyze the in vivo formation of processed asparagine-linked oligosaccharides by developed cells. In addition, developed cells have very low levels of a soluble alpha-mannosidase activity, which is the predominant activity in vegetative cells. This soluble vegetative alpha-mannosidase activity has properties that are reminiscent of the endoplasmic reticulum alpha-mannosidase from rat liver. The intersecting N-acetylglucosaminyltransferase activity that we have described recently in vegetative cells of D. discoideum (Sharkey, D. J., and Kornfeld, R. (1989) J. Biol. Chem. 264, 10411-10419) has a developmental profile that is distinct from that of either of the alpha-mannosidase activities. It has maximum activity at 6 h of development and decreases sharply to its minimum level by 12 h of development. The changes that occur in the levels of these three processing enzymes with development correlate well with the different arrays of asparagine-linked oligosaccharides found in early and late stages of development (Sharkey, D. J., and Kornfeld, R. (1991) J. Biol. Chem. 266, 18485-18497).

Developmental regulation of asparagine-linked oligosaccharide synthesis in Dictyostelium discoideum.

In the preceding report we demonstrated that the expression of two developmentally regulated alpha-mannosidase activities is induced in Dictyostelium discoideum during its differentiation from single-cell amoebae to multicellular organism (Sharkey, D. J., and Kornfeld, R. (1991) J. Biol. Chem. 266, 18477-18484). These activities, designated membrane alpha-mannosidase I (MI) and membrane alpha-mannosidase II (MII), were shown to have several properties in common with rat liver Golgi alpha-mannosidases I and II, respectively, suggesting that MI and MII may play a role in the processing of asparagine-linked oligosaccharides in developing D. discoideum. In this study we analyzed the structures of the asparagine-linked oligosaccharides synthesized by D. discoideum at various stages of development to determine the timing and extent of asparagine-linked oligosaccharide processing. Cells were labeled with [2-3H] mannose, and then total cellular glycoproteins were digested with Pronase to generate glycopeptides that were fractionated on concanavalin A-Sepharose. Glycopeptides from each fraction were digested with endoglycosidase H, both before and after desulfation by solvolysis, and the released, neutral oligosaccharides were sized by high pressure liquid chromatography. At early stages of development, D. discoideum contain predominantly large high mannose-type oligosaccharides (Man9GlcNAc and Man8GlcNAc). Some of these are modified by GlcNAc residues attached beta 1-4 to the mannose-linked alpha 1-6 to the beta-linked core mannose (the "intersecting" position), as well as by fucose, sulfate, and phosphate. In contrast, the oligosaccharides found at late stages of development (18-24 h) have an array of sizes from Man9GlcNAc to Man3GlcNAc. These are still modified by GlcNAc, fucose, sulfate, and phosphate, but the percent of larger high mannose oligosaccharides that are modified with GlcNAc in the intersecting position decreases after 6 h of development, in parallel with the decrease in the intersecting GlcNAc transferase activity. Similarly, the changes in the size of asparagine-linked oligosaccharides synthesized during development correlate well with the appearance of MI and MII activities and suggest that these developmentally regulated alpha-mannosidase activities function in the processing of these oligosaccharides. This is supported further by the observation that oligosaccharide processing was inhibited in late stage cells labeled in the presence of either deoxymannojirimycin, an inhibitor of MI, or swainsonine, an inhibitor of MII.

Identification of an N-acetylglucosaminyltransferase in Dictyostelium discoideum that transfers an "intersecting" N-acetylglucosamine residue to high mannose oligosaccharides.

Glycoproteins synthesized by the cellular slime mold Dictyostelium discoideum have been shown to contain asparagine-linked high-mannose oligosaccharides which have an N-acetylglucosamine group in a novel intersecting position (attached beta 1-4 to the mannose linked alpha 1-6 to the core mannose). We have used crude membrane preparations from vegetative D. discoideum (strain M4) to characterize the enzyme activity responsible for catalyzing the transfer of GlcNAc to the intersecting position of high-mannose oligosaccharides. UDP-GlcNAc:oligosaccharide beta-N-acetylglucosaminyltransferase activity in these preparations attaches GlcNAc to the mannose residue-linked alpha 1-6 to the beta-linked core mannose of the following Man9GlcNAc oligosaccharide as shown by the arrow. (formula; see text) It will also attach GlcNAc to the same intersecting position and/or to the bisecting position (beta-linked core mannose) of the following Man5GlcNAc oligosaccharide. (formula; see text) An analysis of the pH profiles, effects of heat denaturation, and substrate inhibitions on the addition of GlcNAc to either the intersecting or bisecting position of this Man5GlcNAc oligosaccharide indicates that a single enzyme activity is responsible for transferring GlcNAc to both positions. Various oligosaccharides were assayed to determine the substrate specificity of the transferase activity. These data indicate that both the mannose-attached alpha 1-3 and the mannose-attached alpha 1-6 to the mannose receiving the GlcNAc play a critical role in substrate suitability; absence of the alpha 1-6 mannose results in at least a 90% decrease in activity, while absence of the alpha 1-3 mannose results in a completely inactive substrate. This suggests that the minimal substrate is the disaccharide Man alpha 1-3Man.

The role of semen in induction of maternal immune tolerance to pregnancy.

Successful pregnancy requires a state of maternal immune 'tolerance' to accommodate antigens expressed by the conceptus. Implantation failure and placental pathologies largely reflect insufficiencies in maternal immune adaptation, but progress in devising therapeutic strategies to treat these conditions is stalled because the mechanisms underlying the induction and maintenance of maternal tolerance are unknown. Increasingly, clinical and experimental data support the proposal that insemination has consequences for the reproductive process beyond delivery of male gametes. An emerging hypothesis, based mainly on clinical observations and experiments in mice, is that insemination is causally linked to the activation and expansion of populations of lymphocytes mediating forms of 'active' immune tolerance in the implantation site. This review examines existing evidence for a role for semen in the immunology of pregnancy, highlighting the limitations of our existing knowledge and the prospects for future research and its clinical application.

Characterization of crystals of the thermostable DNA polymerase I from Thermus aquaticus.

Thermus aquaticus DNA polymerase I is an enzyme that is of both physiological and technological interest. It carries out template-directed polymerization of DNA at elevated temperatures and is widely used in polymerase chain reaction (PCR). We have obtained crystals of the enzyme that diffracts X-rays to at least 3.0 A resolution in a cubic space group. Determination of the three-dimensional structure of the native enzyme along with those of relevant complexes will greatly enhance our knowledge of molecular events involved in DNA replication, will permit improvements in PCR, and will add to our knowledge of the structural bases of thermostability in proteins.

Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: the Fab is directed against an intermediate in the helix-coil dynamics of the enzyme.

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a gamma turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme. The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme reveal the structural basis of its preference for binding to DNA polymerases of the Thermus species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies remarkably high relative mobility between these two domains.

Structural studies on an inhibitory antibody against Thermus aquaticus DNA polymerase suggest mode of inhibition.

TP7, an antibody against Thermus aquaticus DNA polymerase I (TaqP), is used as a thermolabile switch in 'hot start' variations of PCR to minimize non-specific amplification events. Earlier studies have established that TP7 binds to the polymerase domain of TaqP, competes with primer template complex for binding and is a potent inhibitor of the polymerase activity of TaqP. We report crystallographic determination of the structure of an Fab fragment of TP7 and computational docking of the structure with the known three-dimensional structure of the enzyme. Our observations strongly suggest that the origin of inhibitory ability of TP7 is its binding to enzyme residues involved in DNA binding and polymerization mechanism. Although criteria unbiased by extant biochemical data have been used in identification of a putative solution, the resulting complex offers an eminently plausible structural explanation of biochemical observations. The results presented are of general significance for interpretation of docking experiments and in design of small molecular inhibitors of TaqP, that are not structurally similar to substrates, for use in PCR. Structural and functional similarities noted among DNA polymerases, and the fact that several DNA polymerases are pharmacological targets, make discovery of non-substrate based inhibitors of additional importance.