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

Abstract

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 ...

In Vivo Biosynthetic Studies of the Dolichos biflorus Seed Lectin

“…The inhibition of glycosylation of the subunit precursors with tunicamycin is in agreement with evidence from previous in vitro biosynthetic studies that showed glycosylation to be a cotranslational event in the synthesis of this lectin (27). Such cotranslational glycosylation and subsequent posttranslational alteration of carbohydrate units have been found to be common features in the biosynthesis of many plant lectins (10,12).…”

“…This finding, coupled with structural studies on the subunits (7,8,15,25,26) and the finding of only a single seed lectin mRNA and in vitro translation product (27), suggest that the precursor for subunit II may arise from the precursor for subunit I by carboxyl terminal proteolytic cleavage. Posttranslational proteolytic modifications have been found to occur during the biosynthesis of a number of seed lectins and storage proteins, however such processing events are usually associated with the protein bodies (10).…”

“…It is a tetrameric glycoprotein containing equal amounts of two different subunits, I and II, that have been found to differ only in the length of their carboxyl termini (6-8, 15, 25). Only a single mRNA and in vitro mRNA translation product have been found for this lectin (27). Sequence analysis of the lectin cDNA has shown that it contains the carboxyl terminal sequences of both subunits, suggesting that subunit II may be derived from subunit I by the removal of a 10 amino acid carboxyl terminal segment (26).…”

The unique enzymatic function of field bean (Dolichos lablab) d-galactose specific lectin: a polyphenol oxidase

“…Maximum identity (93%) was observed with a group of uncharacterized legume lectins from Phaseolus filiformus and Phaseolus parvulus. The list includes the well studied DBL and DB58 of Dolichos biflorus [22].…”

Conserved amino acid sequences among plant proteins sorted to protein bodies and plant vacuoles

“…Consistent with such a hypothesis is the fact that these homologous sequences map at the surfaces of the molecules of proteins for which three-dimensional structures are known. [36], Dolichos seed lectin (DBS) [37], Dolichos cell-wall-associated lectin (DB58) [38], Diocleu lectin [39], Lathyrus ochrus lectin [40], arcelin 2 [41], soybean Kunitz trypsin inhibitor [42], Erytrina Kunitz-type trypsin inhibitor [43], winged bean Kunitz-type trypsin inhibitor [44], alfalfa wound-induced trypsin inhibitor [45], tomato protease inhibitor I1 [46], potato protease inhibitor I1 [47], carboxypeptidase Y [7], large T antigen of simian virus 40 [48], pBR322 8-lactamase [49].…”