Susan K. Boehlein

A shrunken-2 Transgene Increases Maize Yield by Acting in Maternal Tissues to Increase the Frequency of Seed Development

This work examines the function of a maize heat-stable, less inhibitor–sensitive form of ADP-glucose pyrophosphorylase, which increases maize yield by increasing seed number. This work shows that this increase requires high temperature during early seed development and results from transgene function in maternal tissues to increase the probability that an ovary will produce a seed. The maize (Zea mays) shrunken-2 (Sh2) gene encodes the large subunit of the rate-limiting starch biosynthetic enzyme, ADP-glucose pyrophosphorylase. Expression of a transgenic form of the enzyme with enhanced heat stability and reduced phosphate inhibition increased maize yield up to 64%. The extent of the yield increase is dependent on temperatures during the first 4 d post pollination, and yield is increased if average daily high temperatures exceed 33°C. As found in wheat (Triticum aestivum) and rice (Oryza sativa), this transgene increases maize yield by increasing seed number. This result was surprising, since an entire series of historic observations at the whole-plant, enzyme, gene, and physiological levels pointed to Sh2 playing an important role only in the endosperm. Here, we present several lines of evidence that lead to the conclusion that the Sh2 transgene functions in maternal tissue to increase seed number and, in turn, yield. Furthermore, the transgene does not increase ovary number; rather, it increases the probability that a seed will develop. Surprisingly, the number of fully developed seeds is only ∼50% of the number of ovaries in wild-type maize. This suggests that increasing the frequency of seed development is a feasible agricultural target, especially under conditions of elevated temperatures.

p37 Induces tumor invasiveness.

Previous studies have shown a statistically significant correlation between human carcinomas and monoclonal antibody detection of a Mycoplasma hyorhinis–encoded protein known as p37. A potential mechanism of p37 is that it might promote invasion and metastasis. Recombinant p37 enhanced the invasiveness of two prostate carcinoma and two melanoma cell lines in a dose-dependent manner in vitro, but did not have a significant effect on tumor cell growth. Furthermore, the increased binding to cell surfaces and the enhanced invasive potential of cancer cells from exposure to p37 could be completely reversed by preincubation of the cancer cells with an anti-p37 monoclonal antibody. Sequence comparisons, followed by three-dimensional molecular modeling, revealed a region of similarity between p37 and influenza hemagglutinin A, a sialic acid–binding protein that plays a critical role in viral entry. Binding of p37 to prostate carcinoma cells was found to be at least partially sialic acid dependent because neuraminidase treatment decreased this binding. Taken together, these observations suggest that M. hyorhinis can infect humans and may facilitate tumor invasiveness via p37. These results further suggest that p37 may be a molecular target for cancer therapy.

Purification and Characterization of Adenosine Diphosphate Glucose Pyrophosphorylase from Maize/Potato Mosaics

Adenosine diphosphate glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in starch biosynthesis. The reaction produces ADP-glucose and pyrophosphate from glucose-1-P and ATP. Investigations from a number of laboratories have shown that alterations in allosteric properties as well as heat stability of this enzyme have dramatic positive effects on starch synthesis in the potato (Solanum tuberosum) tuber and seeds of important cereals. Here, we report the characterization of purified recombinant mosaic AGPases derived from protein motifs normally expressed in the maize (Zea mays) endosperm and the potato tuber. These exhibit properties that should be advantageous when expressed in plants. We also present an in-depth characterization of the kinetic and allosteric properties of these purified recombinant AGPases. These data point to previously unrecognized roles for known allosteric effectors.

Heat stability of maize endosperm ADP-glucose pyrophosphorylase is enhanced by insertion of a cysteine in the N terminus of the small subunit.

ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme in starch biosynthesis. However, plant AGPases differ in several parameters, including spatial and temporal expression, allosteric regulation, and heat stability. AGPases of cereal endosperms are heat labile, while those in other tissues, such as the potato (Solanum tuberosum) tuber, are heat stable. Sequence comparisons of heat-stable and heat-labile AGPases identified an N-terminal motif unique to heat-stable enzymes. Insertion of this motif into recombinant maize (Zea mays) endosperm AGPase increased the half-life at 58°C more than 70-fold. Km values for physiological substrates were unaffected, although Kcat was doubled. A cysteine within the inserted motif gives rise to small subunit homodimers not found in the wild-type maize enzyme. Placement of this N-terminal motif into a mosaic small subunit containing the N terminus from maize endosperm and the C terminus from potato tuber AGPase increases heat stability more than 300-fold.

Heat Stability and Allosteric Properties of the Maize Endosperm ADP-Glucose Pyrophosphorylase Are Intimately Intertwined

ADP-glucose (Glc) pyrophosphorylase (AGPase), a key regulatory enzyme in starch biosynthesis, is highly regulated. Transgenic approaches in four plant species showed that alterations in either thermal stability or allosteric modulation increase starch synthesis. Here, we show that the classic regulators 3-phosphoglyceric acid (3-PGA) and inorganic phosphate (Pi) stabilize maize (Zea mays) endosperm AGPase to thermal inactivation. In addition, we show that glycerol phosphate and ribose-5-P increase the catalytic activity of maize AGPase to the same extent as the activator 3-PGA, albeit with higher Ka (activation constant) values. Activation by fructose-6-P and Glc-6-P is comparable to that of 3-PGA. The reactants ATP and ADP-Glc, but not Glc-1-P and pyrophosphate, protect AGPase from thermal inactivation, a result consistent with the ordered kinetic mechanism reported for other AGPases. 3-PGA acts synergistically with both ATP and ADP-Glc in heat protection, decreasing the substrate concentration needed for protection and increasing the extent of protection. Characterization of a series of activators and inhibitors suggests that they all bind at the same site or at mutually exclusive sites. Pi, the classic “inhibitor” of AGPase, binds to the enzyme in the absence of other metabolites, as determined by thermal protections experiments, but does not inhibit activity. Rather, Pi acts by displacing bound activators and returning the enzyme to its activity in their absence. Finally, we show from thermal inactivation studies that the enzyme exists in two forms that have significantly different stabilities and do not interconvert rapidly.

Detection of antibodies directed at M. hyorhinis p37 in the serum of men with newly diagnosed prostate cancer

BackgroundRecent epidemiologic, genetic, and molecular studies suggest infection and inflammation initiate certain cancers, including cancers of the prostate. Over the past several years, our group has been studying how mycoplasmas could possibly initiate and propagate cancers of the prostate. Specifically, Mycoplasma hyorhinis encoded protein p37 was found to promote invasion of prostate cancer cells and cause changes in growth, morphology and gene expression of these cells to a more aggressive phenotype. Moreover, we found that chronic exposure of benign human prostate cells to M. hyorhinis resulted in significant phenotypic and karyotypic changes that ultimately resulted in the malignant transformation of the benign cells. In this study, we set out to investigate another potential link between mycoplasma and human prostate cancer.MethodsWe report the incidence of men with prostate cancer and benign prostatic hyperplasia (BPH) being seropositive for M. hyorhinis. Antibodies to M. hyorhinis were surveyed by a novel indirect enzyme-linked immunosorbent assay (ELISA) in serum samples collected from men presenting to an outpatient Urology clinic for BPH (N = 105) or prostate cancer (N = 114) from 2006-2009.ResultsA seropositive rate of 36% in men with BPH and 52% in men with prostate cancer was reported, thus leading us to speculate a possible connection between M. hyorhinis exposure with prostate cancer.ConclusionsThese results further support a potential exacerbating role for mycoplasma in the development of prostate cancer.

Probing Allosteric Binding Sites of the Maize Endosperm ADP-Glucose Pyrophosphorylase

Maize (Zea mays) endosperm ADP-glucose pyrophosphorylase (AGPase) is a highly regulated enzyme that catalyzes the rate-limiting step in starch biosynthesis. Although the structure of the heterotetrameric maize endosperm AGPase remains unsolved, structures of a nonnative, low-activity form of the potato tuber (Solanum tuberosum) AGPase (small subunit homotetramer) reported previously by others revealed that several sulfate ions bind to each enzyme. These sites are also believed to interact with allosteric regulators such as inorganic phosphate and 3-phosphoglycerate (3-PGA). Several arginine (Arg) side chains contact the bound sulfate ions in the potato structure and likely play important roles in allosteric effector binding. Alanine-scanning mutagenesis was applied to the corresponding Arg residues in both the small and large subunits of maize endosperm AGPase to determine their roles in allosteric regulation and thermal stability. Steady-state kinetic and regulatory parameters were measured for each mutant. All of the Arg mutants examined—in both the small and large subunits—bound 3-PGA more weakly than the wild type (A50 increased by 3.5- to 20-fold). By contrast, the binding of two other maize AGPase allosteric activators (fructose-6-phosphate and glucose-6-phosphate) did not always mimic the changes observed for 3-PGA. In fact, compared to 3-PGA, fructose-6-phosphate is a more efficient activator in two of the Arg mutants. Phosphate binding was also affected by Arg substitutions. The combined data support a model for the binding interactions associated with 3-PGA in which allosteric activators and inorganic phosphate compete directly.

Exogenous mycoplasmal p37 protein alters gene expression, growth and morphology of prostate cancer cells

We previously showed that the Mycoplasma hyorhinis-encoded protein p37 can promote invasion of cancer cells in a dose-dependent manner, an effect that was blocked by monoclonal antibodies specific for p37. In this study, we further elucidated changes in growth, morphology and gene expression in prostate cancer cell lines when treated with exogenous p37 protein. Incubation with recombinant p37 caused significant nuclear enlargement, denoting active, anaplastic cells and increased the migratory potential of both PC-3 and DU145 cells. Microarray analysis of p37-treated and untreated cells identified eight gene expression clusters that could be broadly classified into three basic patterns. These were an increase in both cell lines, a decrease in either cell line or a cell line-specific differential trend. The most represented functional gene categories included cell cycle, signal transduction and metabolic factors. Taken together, these observations suggest that p37 potentiates the aggressiveness of prostate cancer and thus molecular events triggered by p37 maybe target for therapy.

Structural Insights into the Extracytoplasmic Thiamine-Binding Lipoprotein p37 of Mycoplasma hyorhinis

The Mycoplasma hyorhinis protein p37 has been implicated in tumorigenic transformation for more than 20 years. Though there are many speculations as to its function, based solely on sequence homology, the issue has remained unresolved. Presented here is the 1.6-A-resolution refined crystal structure of M. hyorhinis p37, renamed the extracytoplasmic thiamine-binding lipoprotein (Cypl). The structure shows thiamine pyrophosphate (TPP) and two calcium ions are bound to Cypl and give the first insights into possible functions of the Cypl-like family of proteins. Sequence alignments of Cypl-like proteins between several different species of mycoplasma show that the thiamine-binding site is likely conserved and structural alignments reveal the similarity of Cypl to various binding proteins. While the experimentally determined function of Cypl remains unknown, the structure shows that the protein is a TPP-binding protein, opening up many avenues for future mechanistic studies and making Cypl a possible target for combating mycoplasma infections and tumorigenic transformation.

Characterization of an Autonomously Activated Plant ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch biosynthesis in plants and changes in its catalytic and/or allosteric properties can lead to increased starch production. Recently, a maize (Zea mays)/potato (Solanum tuberosum) small subunit mosaic, MP [Mos(1–198)], containing the first 198 amino acids of the small subunit of the maize endosperm enzyme and the last 277 amino acids from the potato tuber enzyme, was expressed with the maize endosperm large subunit and was reported to have favorable kinetic and allosteric properties. Here, we show that this mosaic, in the absence of activator, performs like a wild-type AGPase that is partially activated with 3-phosphoglyceric acid (3-PGA). In the presence of 3-PGA, enzyme properties of Mos(1–198)/SH2 are quite similar to those of the wild-type maize enzyme. In the absence of 3-PGA, however, the mosaic enzyme exhibits greater activity, higher affinity for the substrates, and partial inactivation by inorganic phosphate. The Mos(1–198)/SH2 enzyme is also more stable to heat inactivation. The different properties of this protein were mapped using various mosaics containing smaller portions of the potato small subunit. Enhanced heat stability of Mos(1–198) was shown to originate from five potato-derived amino acids between 322 and 377. These amino acids were shown previously to be important in small subunit/large subunit interactions. These five potato-derived amino acids plus other potato-derived amino acids distributed throughout the carboxyl-terminal portion of the protein are required for the enhanced catalytic and allosteric properties exhibited by Mos(1–198)/SH2.