Subcellular Localizations of Two <i>Dolichos biflorus</i> Lectins

Etzler, Marilynn E.; Macmillan, S. E.; Scates, S M; Gibson, Donna M.; James, Douglas W.; Cole, D. M.; Thayer, Susan S.

doi:10.1104/pp.76.4.871

Cited by 35 publications

(28 citation statements)

References 30 publications

(21 reference statements)

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“…The best-studied lectins are found in the seeds of leguminous plants, where they may constitute up to 10% of the soluble protein [3,41. Although these lectins exhibit a variety of carbohydrate-binding specificities, striking homologies are apparent when their primary structures are compared [5].…”

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cDNA cloning and in vitro synthesis of the Dolichos biflorus seed lectin

Schnell

Alexander²,

Williams³

et al. 1987

European Journal of Biochemistry

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The Dolichos bzflorus seed lectin contains two structurally related subunits. A cDNA library was constructed using RNA isolated from D. bijlorus seeds actively synthesizing the seed lectin. The library was expressed in Escherichia coli using a lambda Charon 16 vector, and lectin-specific antiserum was used to isolate a seed lectin cDNA. Hybridization of the D. bzflorus seed lectin cDNA to RNA isolated from seeds actively producing both lectin subunits identifies a single-size RNA of 1100 bases. An oligodeoxyribonucleotide probe, constructed from an amino acid sequence common to both lectin subunits, detects the same size RNA. Translation of seed mRNA in vitro and immunoprecipitation of translation products using a lectin-specific antiserum yields a single polypeptide of slightly higher molecular mass than the largest seed lectin subunit. This seed lectin precursor is indistinguishable from a polypeptide synthesized from mRNA hybrid selected by the seed lectin cDNA. These data support the existence of a single polypeptide precursor for both subunit types of the D. biflorus seed lectin and suggest that differences between the subunit types arise by posttranslational processing.Many species of prokaryotic and eukaryotic organisms contain carbohydrate-binding proteins called lectins (for recent reviews see [I, 21). The best-studied lectins are found in the seeds of leguminous plants, where they may constitute up to 10% of the soluble protein [3, 41. Although these lectins exhibit a variety of carbohydrate-binding specificities, striking homologies are apparent when their primary structures are compared [5]. Recent biosynthetic studies show that the subunits of leguminous seed lectins may be encoded by separate genes [6] or may arise by proteolytic splitting of a single polypeptide precursor [7-101. In addition, studies on the biosynthesis of concanavalin A suggest that the subunits of this lectin may arise by cleavage and religation of a single polypeptide chain [ l l , 121. It has become clear from these studies that marked differences exist among the biosynthetic pathways of the closely related leguminous seed lectins. Further definition of these pathways will supply valuable information on the diversity and natural biological roles of these molecules.The Dolichos bijlorus seed lectin ( M , = 110000) exhibits a high degree of specificity for N-acetylgalactosamine [13, 141. This lectin is a glycoprotein tetramer composed of two types of subunits (subunit I, M , = 27700; subunit 11, M , = 27300) with an apparent 2: 2 subunit stoichiometry [15,16]. Extensive structural analysis of the D. bzjlorus lectin subunits indicates that the two subunit types are nearly identical except for their carboxyl-terminal amino acid residues [17, 181. Despite this remarkable homology, only the larger subunit, subunit I, has the ability to bind carbohydrate [19].In order more clearly to define the relationships and understand the origins of the D. bijlorus seed lectin subunits, we have studied the synthesis and processing of the lectin ...

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cDNA cloning and in vitro synthesis of the Dolichos biflorus seed lectin

Schnell

Alexander²,

Williams³

et al. 1987

European Journal of Biochemistry

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“…The pellets were then incubated overnight at 4°C in 0.01 M phosphate (pH 7.2) containing 1.0 M NaCl, 0.1 mg/mL PMSF, and 0.02% NaN3. Alternatively, the pellets were digested with cellulase and pectinase as previously described (10) to extract the remaining proteins. These treatments have previously been shown to be effective in removing DB58 that is noncovalently associated with the cell walls (10).…”

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“…Antisera to the denatured seed lectin and DB57 were prepared according to standard procedures used in our laboratory (24). The D 5 cell suspension cultures, in the middle of the logarithmic growth phase, were homogenized in a BeadBeater (Biospec Products) using 0.5 mm diameter beads in the presence of a previously described extraction buffer (10,25). The supernatant of this extract was subjected to SDS-urea PAGE, transferred to nitrocellulose, and probed with antiserum against the denatured seed lectin.…”

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Heath Shock Enhances the Synthesis of a Lectin-Related Protein in Dolichos biflorus Cell Suspension Cultures

Spadoro-Tank¹,

Etzler²

1988

Plant Physiol.

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Dolichos biflorus cell suspension cultures synthesize at least two lectins or lectin-like proteins that are related to the seed lectin from this plant. The synthesis of one of these proteins, DB57, is greatly enhanced in response to heat shock; this enhancement of synthesis is dependent upon the growth phase of the cells in culture. Early in the growth curve, when the cells are in lag phase, there is no detectable induction of DB57 synthesis above the levels found in control cells. Enhancement of DB57 synthesis becomes apparent during the last half of the exponential phase of growth at which time there is approximately a 10-fold increase in the amount of newly synthesized DB57 at 42°C compared with the amount of DB57 synthesized at the control temperature of 27°C. The implication of this finding is discussed with respect to the role of lectins in plants.The vegetative-tissues of the plant Dolichos biflorus contain at least four lectins or lectin-like proteins which are related to the seed lectin from this plant (11). DB58, formerly called CRM, is the most well-characterized of these lectins (25); it was first detected in the stems and leaves of the plant by its cross-reaction with antiserum against the seed lectin (24). Three other proteins, DB57, DB50, and DB46, were detected in extracts of stems and leaves by chromatography on blood group A+H Sepharose (8,11,22); DB46 has also been isolated from roots (22). Unlike DB58, these proteins in their native forms do not cross-react with antiserum against the native seed lectin or DB58; upon denaturation, however, they do react with these antisera and with antiserum prepared against denatured seed lectin (1 1). This cross-reactivity and the ability of the proteins to bind to blood group A+H substance suggests that they are related to the blood group A specific seed lectin (9, 1 1).Several observations during the course of our work suggested that the synthesis of at least one of these lectins may be induced in response to stress (5 At present, little is known about the identity of the heat shock proteins in plants. In soybean seedlings, it has been reported that temperature stress results in the enhanced synthesis of the 11 S and 7S storage proteins (18). In addition, it has recently been reported that the synthesis of phytohemagglutinin is enhanced during temperature stress in Phaseolus vulgaris cotyledons (3).In the present study, D. biflorus cell suspension cultures were used as an experimental system in which to examine the effects of heat shock on lectin synthesis. We demonstrate that the synthesis of the lectin-related protein, DB57, is greatly enhanced in response to heat shock and that this induction is dependent upon the growth phase of the cells in culture.MATERIALS AND METHODS Plant Cell Cultures. Dolichos biflorus cell suspension cultures, established from callus cultures, were maintained in hormone free media as previously described (13) PMSF.3 After centrifugation at 27,000g, the pellets were washed at 4°C for 5 min with PBS containing 0.1 mg/mL...

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“…Nonseed legume lectins have been reported to be localized in either the cell wall (4,14) or in vacuoles of roots (19) and in protein storage vacuoles of leaves and bark (9). The intracellular distribution of lectin-like proteins arcelin and amylase inhibitor has not been determined although it is likely that these will also prove to be vacuolar proteins.…”

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The Purification, Properties, and Localization of an Abundant Legume Seed Lectin Cross-Reactive Material from Spartium junceum

et al. 1991

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The seeds of Spartium junceum contained a large quantity of lectin-like protein that did not appear to be either a hemagglutinin or active lectin. The cross-reactive material (CRM), like most legume seed lectins, was a tetrameric glycoprotein of about 130,000 M,. The singlesized subunits of about 33,000 Mr were not covalently associated. The amino acid composition was typical of legume lectins and was rich in hydroxy-amino acids and poor in sulfur-containing amino acids. The Spartium CRM contained about 3.5% covalently associated carbohydrate, most likely of the high-mannose type, since the CRM was precipitated by concanavalin A. The CRM was localized by electron-microscopic immunocytochemistry and found to be exclusively in protein-filled vacuoles (protein bodies). Because this protein was so similar immunologically, structurally, and in its physiology, to classic legume seed lectins, it is most likely a lectin homolog. Similar seed lectin CRMs appear to be both common and widespread in the Leguminosae.Although the seeds of many legume species contain large quantities of lectin, there appears to be an equal number of legume species that are devoid of lectin activity (15,17). These seed lectin "negative" legume species, however, in many cases, contain lectin-like proteins-that is, proteins that are immunologically cross-reactive with legume lectins.Even though seed lectin CRMs3 are very widely distributed within the legume family and often appear to be abundant seed proteins, as far as we know, none has been purified and characterized. Whereas lectin CRMs from other plant tissues such as stems, leaves, and roots, have been studied, these CRMs generally appear to be present in very low amounts, and even though closely related to seed lectins, they often possess significantly different structures. We believe that the low-abundance, apparently non-vacuolar, lectin CRMs, perhaps typified by those from Dolichos (2, 16) and peas (1), are functionally distinct from the high-abundance vacuolar local-'The electron microscope facilities were made available in part through Public Health Service Grant S-507-RRO7010-13. This research was supported by National Science Foundation Grant DMB 8517499. 'Abbreviation: CRM, cross-reactive material.ized lectins that have been found in many legume seeds, as well as in bark (5, 1 1) and leaves (6). We wondered whether the seed lectin CRMs that we had previously observed were forms of seed lectins or similar to other lectin CRMs that have been described or perhaps if they might be a new class of lectin-related protein. For this reason, we have begun to study these proteins in more detail.In the study reported here, we employed Western blots to show the presence of lectin CRMs in numerous nonhemagglutinating legume seed extracts and have described the purification, localization, and some properties ofa specific lectin CRM from the seeds of the legume Spartium junceum. MATERIALS AND METHODSHemagglutinin assays, enzyme assays, and Ouchterlony double diffusion were as described prev...

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Subcellular Localizations of Two Dolichos biflorus Lectins

Cited by 35 publications

References 30 publications

cDNA cloning and in vitro synthesis of the Dolichos biflorus seed lectin

cDNA cloning and in vitro synthesis of the Dolichos biflorus seed lectin

Heath Shock Enhances the Synthesis of a Lectin-Related Protein in Dolichos biflorus Cell Suspension Cultures

The Purification, Properties, and Localization of an Abundant Legume Seed Lectin Cross-Reactive Material from Spartium junceum

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