Molecular characterization of the mycobacterial heparin-binding hemagglutinin, a mycobacterial adhesin

Although it generally is accepted that the interaction of Mycobacterium tuberculosis with alveolar macrophages is a key step in the pathogenesis of tuberculosis, interactions with other cell types, especially epithelial cells, also may be important. In this study we describe the molecular characterization of a mycobacterial heparin-binding hemagglutinin (HBHA), a protein that functions as an adhesin for epithelial cells. The structural gene was cloned from M. tuberculosis and bacillus Calmette–Guérin, and the sequence was found to be identical between the two species. The calculated Mr was smaller than the observed Mr when analyzed by SDS/PAGE. This difference can be attributed to the Lys/Pro-rich repeats that occur at the C-terminal end of the protein and to a putative carbohydrate moiety. Glycosylation of HBHA appears to protect the protein from proteolytic degradation, which results in the removal of the C-terminal Lys/Pro-rich region responsible for binding of HBHA to sulfated carbohydrates. Evidence suggests that glycosylation is also important for HBHA-mediated hemagglutination and for certain immunologic properties of the protein. Finally, the absence of a signal peptide in the coding region of HBHA raises the possibility that this protein is not secreted via the general secretion pathway.

The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein– protein interactions independent of carbohydrate moiety

The lecticans are a family of chondroitin sulfate proteoglycans including aggrecan, versican, neurocan, and brevican. The C-terminal globular domains of lecticans are structurally related to selectins, consisting of a C-type lectin domain flanked by epidermal growth factor and complement regulatory protein domains. The C-type lectin domain of versican has been shown to bind tenascin-R, an extracellular matrix protein specifically expressed in the nervous system, and the interaction was presumed to be mediated by a carbohydrate–protein interaction. In this paper, we show that the C-type lectin domain of brevican, another lectican that is specifically expressed in the nervous system, also binds tenascin-R. Surprisingly, this interaction is mediated by a protein–protein interaction through the fibronectin type III domains 3–5 of tenascin-R, independent of any carbohydrates or sulfated amino acids. The lectin domains of versican and other lecticans also bind the same domain of tenascin-R by protein–protein interactions. Surface plasmon resonance analysis revealed that brevican lectin has at least a 10-fold higher affinity than the other lectican lectins. Tenascin-R is coprecipitated with brevican from adult rat brain extracts, suggesting that tenascin-R and brevican form complexes in vivo. These results demonstrate that the C-type lectin domain can interact with fibronectin type III domains through protein–protein interactions, and suggest that brevican is a physiological tenascin-R ligand in the adult brain.

Neuroprotective activity of a new class of steroidal inhibitors of the N-methyl-d-aspartate receptor

Release of the excitatory neurotransmitter glutamate and the excessive stimulation of N-methyl-d-aspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3α-ol-5β-pregnan-20-one hemisuccinate (3α5βHS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3α5βHS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3α5βHS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The in vitro and in vivo neuroprotective effects of 3α5βHS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.

Electrophysiological studies on rat dorsal root ganglion neurons after peripheral axotomy: Changes in responses to neuropeptides

The effect of three peptides, galanin, sulfated cholecystokinin octapeptide, and neurotensin (NT), was studied on acutely extirpated rat dorsal root ganglia (DRGs) in vitro with intracellular recording techniques. Both normal and peripherally axotomized DRGs were analyzed, and recordings were made from C-type (small) and A-type (large) neurons. Galanin and sulfated cholecystokinin octapeptide, with one exception, had no effect on normal C- and A-type neurons but caused an inward current in both types of neurons after sciatic nerve cut. In normal rats, NT caused an outward current in C-type neurons and an inward current in A-type neurons. After sciatic nerve cut, NT only caused an inward current in both C- and A-type neurons. These results suggest that (i) normal DRG neurons express receptors on their soma for some but not all peptides studied, (ii) C- and A-type neurons can have different types of receptors, and (iii) peripheral nerve injury can change the receptor phenotype of both C- and A-type neurons and may have differential effects on these neuron types.

Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti

Succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti, is composed of polymerized octasaccharide subunits, each of which consists of one galactose and seven glucoses with succinyl, acetyl, and pyruvyl modifications. Production of specific low molecular weight forms of R. meliloti exported and surface polysaccharides, including succinoglycan, appears to be important for nodule invasion. In a previous study of the roles of the various exo gene products in succinoglycan biosynthesis, exoP, exoQ, and exoT mutants were found to synthesize undecaprenol-linked fully modified succinoglycan octasaccharide subunits, suggesting possible roles for their gene products in polymerization or transport. Using improved techniques for analyzing succinoglycan biosynthesis by these mutants, we have obtained evidence indicating that R. meliloti has genetically separable systems for the synthesis of high molecular weight succinoglycan and the synthesis of a specific class of low molecular weight oligosaccharides consisting of dimers and trimers of the octasaccharide subunit. Models to account for our unexpected findings are discussed. Possible roles for the ExoP, ExoQ, and ExoT proteins are compared and contrasted with roles that have been suggested on the basis of homologies to key proteins involved in the biosynthesis of O-antigens and of certain exported or capsular cell surface polysaccharides.

Enhanced Glycosylation and Sulfation of Secretory Proteoglycans Is Coupled to the Expression of a Basic Secretory Protein

We have used coexpression of a salivary basic proline-rich protein (PRP) along with a proline-rich proteoglycan (PRPg) in pituitary AtT-20 cells to examine the regulation of glycosaminoglycan (GAG) biosynthesis and the storage of these secretory products for regulated secretion. The basic PRP caused a dose-dependent increase in sulfation of PRPg and also increased the extent to which PRPg polypeptide backbones are modified by a GAG chain. The sulfation of an endogenous proteoglycan was similarly increased in the presence of basic PRP; however, other sulfated secretory products of AtT-20 cells were unaffected. These results imply that enzymes functioning in elongation and sulfation of proteoglycans are coordinately regulated and that their activities respond to a change in the milieu of the intracellular transport pathway. Analysis of the regulated secretion of both the basic PRP and PRPg has indicated that while the presence of the GAG chain improves the storage of PRPg, the presence of PRPg does not increase the storage of basic PRP. Therefore, sulfation of GAGs does not appear to be a primary factor in regulated secretory sorting.

Distinct Molecular Events during Secretory Granule Biogenesis Revealed by Sensitivities to Brefeldin A

The biogenesis of peptide hormone secretory granules involves a series of sorting, modification, and trafficking steps that initiate in the trans-Golgi and trans-Golgi network (TGN). To investigate their temporal order and interrelationships, we have developed a pulse–chase protocol that follows the synthesis and packaging of a sulfated hormone, pro-opiomelanocortin (POMC). In AtT-20 cells, sulfate is incorporated into POMC predominantly on N-linked endoglycosidase H-resistant oligosaccharides. Subcellular fractionation and pharmacological studies confirm that this sulfation occurs at the trans-Golgi/TGN. Subsequent to sulfation, POMC undergoes a number of molecular events before final storage in dense-core granules. The first step involves the transfer of POMC from the sulfation compartment to a processing compartment (immature secretory granules, ISGs): Inhibiting export of pulse-labeled POMC by brefeldin A (BFA) or a 20°C block prevents its proteolytic conversion to mature adrenocorticotropic hormone. Proteolytic cleavage products were found in vesicular fractions corresponding to ISGs, suggesting that the processing machinery is not appreciably activated until POMC exits the sulfation compartment. A large portion of the labeled hormone is secreted from ISGs as incompletely processed intermediates. This unregulated secretory process occurs only during a limited time window: Granules that have matured for 2 to 3 h exhibit very little unregulated release, as evidenced by the efficient storage of the 15-kDa N-terminal fragment that is generated by a relatively late cleavage event within the maturing granule. The second step of granule biogenesis thus involves two maturation events: proteolytic activation of POMC in ISGs and a transition of the organelle from a state of high unregulated release to one that favors intracellular storage. By using BFA, we show that the two processes occurring in ISGs may be uncoupled: although the unregulated secretion from ISGs is impaired by BFA, proteolytic processing of POMC within this organelle proceeds unaffected. The finding that BFA impairs constitutive secretion from both the TGN and ISGs also suggests that these secretory processes may be related in mechanism. Finally, our data indicate that the unusually high levels of unregulated secretion often associated with endocrine tumors may result, at least in part, from inefficient storage of secretory products at the level of ISGs.

Sequencing of 3-O sulfate containing heparin decasaccharides with a partial antithrombin III binding site

Heparin- and heparan sulfate-like glycosaminoglycans (HLGAGs) represent an important class of molecules that interact with and modulate the activity of growth factors, enzymes, and morphogens. Of the many biological functions for this class of molecules, one of its most important functions is its interaction with antithrombin III (AT-III). AT-III binding to a specific heparin pentasaccharide sequence, containing an unusual 3-O sulfate on a N-sulfated, 6-O sulfated glucosamine, increases 1,000-fold AT-III's ability to inhibit specific proteases in the coagulation cascade. In this manner, HLGAGs play an important biological and pharmacological role in the modulation of blood clotting. Recently, a sequencing methodology was developed to further structure-function relationships of this important class of molecules. This methodology combines a property-encoded nomenclature scheme to handle the large information content (properties) of HLGAGs, with matrix-assisted laser desorption ionization MS and enzymatic and chemical degradation as experimental constraints to rapidly sequence picomole quantities of HLGAG oligosaccharides. Using the above property-encoded nomenclature-matrix-assisted laser desorption ionization approach, we found that the sequence of the decasaccharide used in this study is ΔU2SHNS,6SI2SHNS,6SI2SHNS,6SIHNAc,6SGHNS,3S,6S (±DDD4–7). We confirmed our results by using integral glycan sequencing and one-dimensional proton NMR. Furthermore, we show that this approach is flexible and is able to derive sequence information on an oligosaccharide mixture. Thus, this methodology will make possible both the analysis of other unusual sequences in HLGAGs with important biological activity as well as provide the basis for the structural analysis of these pharamacologically important group of heparin/heparan sulfates.

Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis

Arabidopsis cyt1 mutants have a complex phenotype indicative of a severe defect in cell wall biogenesis. Mutant embryos arrest as wide, heart-shaped structures characterized by ectopic accumulation of callose and the occurrence of incomplete cell walls. Texture and thickness of the cell walls are irregular, and unesterified pectins show an abnormally diffuse distribution. To determine the molecular basis of these defects, we have cloned the CYT1 gene by a map-based approach and found that it encodes mannose-1-phosphate guanylyltransferase. A weak mutation in the same gene, called vtc1, has previously been identified on the basis of ozone sensitivity due to reduced levels of ascorbic acid. Mutant cyt1 embryos are deficient in N-glycosylation and have an altered composition of cell wall polysaccharides. Most notably, they show a 5-fold decrease in cellulose content. Characteristic aspects of the cyt1 phenotype, including radial swelling and accumulation of callose, can be mimicked with the inhibitor of N-glycosylation, tunicamycin. Our results suggest that N-glycosylation is required for cellulose biosynthesis and that a deficiency in this process can account for most phenotypic features of cyt1 embryos.

Biological impact on mineral dissolution: Application of the lichen model to understanding mineral weathering in the rhizosphere

Microorganisms modify rates and mechanisms of chemical and physical weathering and clay growth, thus playing fundamental roles in soil and sediment formation. Because processes in soils are inherently complex and difficult to study, we employ a model based on the lichen–mineral system to identify the fundamental interactions. Fixed carbon released by the photosynthetic symbiont stimulates growth of fungi and other microorganisms. These microorganisms directly or indirectly induce mineral disaggregation, hydration, dissolution, and secondary mineral formation. Model polysaccharides were used to investigate direct mediation of mineral surface reactions by extracellular polymers. Polysaccharides can suppress or enhance rates of chemical weathering by up to three orders of magnitude, depending on the pH, mineral surface structure and composition, and organic functional groups. Mg, Mn, Fe, Al, and Si are redistributed into clays that strongly adsorb ions. Microbes contribute to dissolution of insoluble secondary phosphates, possibly via release of organic acids. These reactions significantly impact soil fertility. Below fungi–mineral interfaces, mineral surfaces are exposed to dissolved metabolic byproducts. Through this indirect process, microorganisms can accelerate mineral dissolution, leading to enhanced porosity and permeability and colonization by microbial communities.

Several Thaumatin-Like Proteins Bind to β-1,3-Glucans1

Pathogenesis-related proteins from intercellular fluid washings of stressed barley (Hordeum vulgare L.) leaves were analyzed to determine their binding to various water-insoluble polysaccharides. Three proteins (19, 16, and 15 kD) bound specifically to several water-insoluble β-1,3-glucans. Binding of the barley proteins to pachyman occurred quickly at 22°C at pH 5.0, even in the presence of 0.5 m NaCl, 0.2 m urea, and 1% (v/v) Triton X-100. Bound barley proteins were released by acidic treatments or by boiling in sodium dodecyl sulfate. Acid-released barley proteins could bind again specifically and singly to pachyman. Water-soluble laminarin and carboxymethyl-pachyman competed for the binding of the barley proteins to pachyman. The N-terminal sequence of the 19-kD barley β-1,3-glucan-binding protein showed near identity to the barley seed protein BP-R and high homology to other thaumatin-like (TL) permatins. The 16-kD barley protein was also homologous to TL proteins, whereas the 15-kD barley protein N-terminal sequence was identical to the pathogenesis-related Hv-1 TL protein. Antifungal barley protein BP-R and corn (Zea mays) zeamatin were isolated by binding to pachyman. Two extracellular proteins from stressed pea (Pisum sativum L.) also bound to pachyman and were homologous to TL proteins.

Changes in Cell Wall Composition during Ripening of Grape Berries1

Cell walls were isolated from the mesocarp of grape (Vitis vinifera L.) berries at developmental stages from before veraison through to the final ripe berry. Fluorescence and light microscopy of intact berries revealed no measurable change in cell wall thickness as the mesocarp cells expanded in the ripening fruit. Isolated walls were analyzed for their protein contents and amino acid compositions, and for changes in the composition and solubility of constituent polysaccharides during development. Increases in protein content after veraison were accompanied by an approximate 3-fold increase in hydroxyproline content. The type I arabinogalactan content of the pectic polysaccharides decreased from approximately 20 mol % of total wall polysaccharides to about 4 mol % of wall polysaccharides during berry development. Galacturonan content increased from 26 to 41 mol % of wall polysaccharides, and the galacturonan appeared to become more soluble as ripening progressed. After an initial decrease in the degree of esterification of pectic polysaccharides, no further changes were observed nor were there large variations in cellulose (30–35 mol % of wall polysaccharides) or xyloglucan (approximately 10 mol % of wall polysaccharides) contents. Overall, the results indicate that no major changes in cell wall polysaccharide composition occurred during softening of ripening grape berries, but that significant modification of specific polysaccharide components were observed, together with large changes in protein composition.

Phosphorylated α(1→4)Glucans as Substrate for Potato Starch-Branching Enzyme I1

The possible involvement of potato (Solanum tuberosum L.) starch-branching enzyme I (PSBE-I) in the in vivo synthesis of phosphorylated amylopectin was investigated in in vitro experiments with isolated PSBE-I using 33P-labeled phosphorylated and 3H end-labeled nonphosphorylated α(1→4)glucans as the substrates. From these radiolabeled substrates PSBE-I was shown to catalyze the formation of dual-labeled (3H/33P) phosphorylated branched polysaccharides with an average degree of polymerization of 80 to 85. The relatively high molecular mass indicated that the product was the result of multiple chain-transfer reactions. The presence of α(1→6) branch points was documented by isoamylase treatment and anion-exchange chromatography. Although the initial steps of the in vivo mechanism responsible for phosphorylation of potato starch remains elusive, the present study demonstrates that the enzyme machinery available in potato has the ability to incorporate phosphorylated α(1→4)glucans into neutral polysaccharides in an interchain catalytic reaction. Potato mini tubers synthesized phosphorylated starch from exogenously supplied 33PO43− and [U-14C]Glc at rates 4 times higher than those previously obtained using tubers from fully grown potato plants. This system was more reproducible compared with soil-grown tubers and was therefore used for preparation of 33P-labeled phosphorylated α(1→4)glucan chains.

Characterization of SU1 Isoamylase, a Determinant of Storage Starch Structure in Maize1

Function of the maize (Zea mays) gene sugary1 (su1) is required for normal starch biosynthesis in endosperm. Homozygous su1- mutant endosperms accumulate a highly branched polysaccharide, phytoglycogen, at the expense of the normal branched component of starch, amylopectin. These data suggest that both branched polysaccharides share a common precursor, and that the product of the su1 gene, designated SU1, participates in kernel starch biosynthesis. SU1 is similar in sequence to α-(1→6) glucan hydrolases (starch-debranching enzymes [DBEs]). Specific antibodies were produced and used to demonstrate that SU1 is a 79-kD protein that accumulates in endosperm coincident with the time of starch biosynthesis. Nearly full-length SU1 was expressed in Escherichia coli and purified to apparent homogeneity. Two biochemical assays confirmed that SU1 hydrolyzes α-(1→6) linkages in branched polysaccharides. Determination of the specific activity of SU1 toward various substrates enabled its classification as an isoamylase. Previous studies had shown, however, that su1- mutant endosperms are deficient in a different type of DBE, a pullulanase (or R enzyme). Immunoblot analyses revealed that both SU1 and a protein detected by antibodies specific for the rice (Oryza sativa) R enzyme are missing from su1- mutant kernels. These data support the hypothesis that DBEs are directly involved in starch biosynthesis.

Cloning and Characterization of AtRGP11 : A Reversibly Autoglycosylated Arabidopsis Protein Implicated in Cell Wall Biosynthesis

A reversibly glycosylated polypeptide from pea (Pisum sativum) is thought to have a role in the biosynthesis of hemicellulosic polysaccharides. We have investigated this hypothesis by isolating a cDNA clone encoding a homolog of Arabidopsis thaliana, Reversibly Glycosylated Polypeptide-1 (AtRGP1), and preparing antibodies against the protein encoded by this gene. Polyclonal antibodies detect homologs in both dicot and monocot species. The patterns of expression and intracellular localization of the protein were examined. AtRGP1 protein and RNA concentration are highest in roots and suspension-cultured cells. Localization of the protein shows it to be mostly soluble but also peripherally associated with membranes. We confirmed that AtRGP1 produced in Escherichia coli could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates. Possible sites for UDP-sugar binding and glycosylation are discussed. Our results are consistent with a role for this reversibly glycosylated polypeptide in cell wall biosynthesis, although its precise role is still unknown.