Hetty M. Stinissen

Sambucus nigra agglutinin is located in protein bodies in the phloem parenchyma of the bark.

The bark of some young woody stems contains storage proteins which are subject to an annual rhythm: they accumulate in the autumn and are mobilized in the spring. We show here that the bark phoem-parenchyma cells of Sambucus nigra L. contain numerous protein bodies, and that the bark lectin (S. nigra agglutinin) which undergoes an annual rhythm is localized in these protein bodies. The protein bodies in the cotyledons of legume seeds also contain lectin, indicating that lectins may be storage compounds themselves or may have a function in storage and-or mobilization processes.

A genetic basis for the origin of six different isolectins in hexaploid wheat.

Wheat (Triticum aestivum) germ agglutinin represents a complex mixture of multiple isolectin forms. Upon ion exchange chromatography at pH 3.8, three isolectins can be separated, each of which is composed of two identical subunits. At pH 5.0, however, three additional isolectins can be distinguished, which are built up of two different subunits (heteromeric lectins). Evidence is presented that these heterodimers are normal constituents of the wheat embryo cells. Analyses of the isolectin patterns in extracts from Triticum monococcum, Triticum turgidum dicoccum and Triticum aestivum, provide evidence that each genome, either in simple or complex (polyploid) genomes, directs the synthesis of a single lectin subunit species. In addition, a comparison of the isolectin pattern in these wheat species of increasing ploidy level, made it possible to determine unequivocally the genome by which the individual lectin subunits in polyploid species are coded for. The possible use of lectins in studies on the origin of individual genoms in polyploid species is discussed.

Lectin synthesis in developing and germinating wheat and rye embryos

Wheat (Triticum aestivum L.) and rye (Secale cereale L.) lectins are specifically synthesized during seed formation. They accumulate exponentially in the primary axes in a period coinciding with the development of this complex organ. Since the specific lectin content also increases dramatically, there is apparently an outburst of lectin synthesis during the development of the primary axes. Germinating embryos also synthesize some lectin. The fortunate availability of a highly specific procedure for the isolation of cereal lectins enabled us to follow the kinetics of their synthesis during early germination. Stored mRNAs appear to be involved in this residual lectin synthesis.

Occurrence and immunological relationships of lectins in gramineous species

A survey of the occurrence of lectins in seeds from more than 100 grass species showed that all species belonging to the Triticeae tribe and the genera Brachypodium and Oryza contain lectins. All these lectins have the same sugar-binding specificity and are related to wheat-germ agglutinin, but to different degrees. Lectins from Triticeae species are immunologically indistinguishable from wheat lectin, whereas Brachypodium and rice lectins are only immunologically related to the wheat lectin. Attempts to detect lectin-deficient lines or varieties in wild and cultivated species of the three lectin-containing groups were unsuccessful. The possible use of lectins as a chemotaxonomic tool is discussed.

Subcellular site of lectin synthesis in developing rice embryos.

Embryos of developing rice (Oryza sativa L. cv. Koshihikari) caryopses which actively synthesize lectin were labelled with [35S]cysteine for different times and newly synthesized rice lectin was isolated by affinity chromatography. Gel filtration of embryo extracts on Sepharose‐4B indicated that a large portion of the labelled lectin was associated with the particulate fraction. Experiments with detergent indicated that this lectin was sequestered within organelles. When extracts of pulse‐labelled embryos were fractionated on isopycnic sucrose gradients, this detergent‐released lectin banded in the same density‐region as the endoplasmic reticulum (ER) marker enzyme NADH‐cytochrome c reductase. Both radioactivity in rice lectin and the enzyme activity shifted towards a higher density in the presence of 2 mM Mg acetate, indicating that the labelled lectin was associated with the rough ER. The ER‐bound lectin could be chased from this organelle when tissue was incubated in unlabelled cysteine following a 1 h pulse of labelled cysteine. Radioactivity chased out of the ER with a half‐life of ˜4 h and accumulated in the soluble fraction. In the ER the lectin was present as a polypeptide with mol. wt. 23 000, while in the soluble fraction it occurred as polypeptides with mol. wt. 18 000, 10 000 and 8000. The rice lectin in the ER is capable of binding carbohydrates since it binds readily to the affinity gels. It is associated into dimers with an approximate mol. wt. of 46 000. The results show that newly synthesized rice lectin is transiently sequestered within the ER before further transport and processing take place.

The Rice Lectin and its Relationship to Cereal Lectins

Summary Rice Oryza sativa embryos contain a N-acetylglucosamine-specific lectin which is exclusively located in the primary axes. Affinity chromatography on immobilized N-acetylglucosamine yielded essentially pure preparations. The molecular structure of the rice lectin has been determined using in vivo labelling experiments. It is composed of two identical subunits of mol.wt. 18,000, which are partially cleaved into two smaller polypeptides of mol.wt. 12,000 and 8,000 respectively. Although rice lectin resembles wheat germ agglutinin ad other cereal lectins in several aspects, there are some differences. Rice lectin does not dissociate at low p H and has a 4-fold higher specific agglutination activity than cereal lectins. In spite of the far taxonomic distance between rice and wheat, their respective lectins are immunologically related, although not identical. The possible use of rice lectin as an alternative to wheat germ agglutinin in affinity chromatography systems for the isolation of glycoproteins or cells is discussed.

Subunit exchange between lectins from different cereal species.

Lectins from Triticum monococcum, Secale cereale (rye), and Hordeum vulgare (barley) can exchange their subunits in vitro and thereby form (intergeneric) heteromeric lectins. An analysis of the isolectin pattern of a Triticale variety revealed that intergeneric heterodimers of wheat and rye lectin subunits are normal constituents of the embryo cells. It appears, therefore, that these different cereal lectins are structurally so closely related that their subunits can not distinguish between identical and nonidentical partners when they associate into dimers.

In vivo synthesis and processing of cereal lectins

The synthesis and processing of cereal lectins was followed in vivo. The initial translation products of lectin genes are higher molecular weight (28 K) precursors, which are post-translationally processed in a single step into authentic lectin polypeptides (23 K). The conversion of precursor into mature product is a rather slow process (the precursor has a half life of 36 min) and is apparently not a prerequisite for biological activity since the precursor exhibits sugar binding activity. Because of the striking resemblances between the processing of cereal lectins and vectorial processing of cytoplasmatically made chloroplast, mitochondrial and glyoxysomal proteins, vectorial processing of cereal lectins might be a means of transporting these proteins through a membrane into an extra-cytoplasmic compartment.

Recent Advances in Biochemistry, Cell Biology, Physiology, Biosynthesis and Genetics of Gramineae Lectins

Summary During the last years, evidence has accumulated that within the grass family only one major type of agglutinin occurs.It can be divided further into three subtypes, which according to the taxonomic devisions where they are found, are designated as cereal, rice and Brachypodium lectins.These three types are both structurally and serologically related to each other, and most probably have a common ancestor. Of special interest are the isolectins in allopolyploid cereals such as tetraploid and hexaploid wheats.In embryo cells of these species, each individual genome directs the synthesis of its own characteristic lectin polypeptide.The occurrence of such multiple molecular lectin forms has been exploited for the chromosomal location of the lectin genes on the homoeologous group 1 chromosomes of wheat and Aegilops umbellulata. Considerable progress has been made also with respect to some typical biological and cell-biological aspects of cereal and rice lectins.It has become evident that, at least the seed lectins, are exclusively located in the primary axes where they appear to be limited to regions that establish contact with the soil during germination.Besides in embryos, Gramineae lectins are found also in tips of roots of young seedlings and adventitious roots of adult plants.Moreover, even leaves of adult barley plants can be a major site of lectin location and synthesis, at least under some (unknown) conditions.At the subcellular level, Gramineae lectins appear to be sequestered in the lumen of protein body-like organelles in embryo cells and in the lumen of vacuoles in cells of adventitious roots.At both sites, the lectins are confined merely to the peripheral regions close to the membrane of these organelles. Recently, important advances have been made with respect to the biosynthesis, processing and intracellular transport of cereal and rice lectins.Both types of lectins are specifically synthesized in developing primary axes, at least with the exception of some residual synthesis during early germination.Moreover, their biosynthesis is promoted by abscisic acid, which might indicate that cereal and rice lectins are dormancy-specific proteins.At the molecular level, the translation products of lectin mRNAs undergo several modifications and are intracellularly transported before the mature lectin products reach their final destination.Both cereal and rice lectins are synthesized as higher Mw precursors (23,000 MW), which are post-translationally converted into 18,000 MW lectin polypeptides.This 18,000 MW protein is the final product of cereal lectin synthesis but is, at least in some rice species, further cleaved into two smaller polypeptides of MW 10,000 and 8,000 respectively.Before the post-translational processing(s) take place, the lectin precursors are transiently associated with the endoplasmic reticulum.It is not clear, however, whether these precursors are transported to other organelles and if so, to what kind of organelles. A tissue-specific cereal lectins occurs in couch grass (Agropyrum repens) leaves.Although this leaf lectin is clearly related to the embryo lectin of the same species it has a slightly higher MW and different sugar-binding and blood-group specificity.Moreover it is serologically different from the embryo lectin. Based on the current knowledge of various aspects of Gramineae lectins two sorts of physiological functions have been proposed.At present, however, it is impossible to decide whether these proteins have a defensive role against pathogenic fungi or fulfill a more specific endogenous function.Also the possibility can not be excluded that they perform various and multiple functions, and that these functions can be influenced by endogenous and/or exogenous factors.

Two-step processing of in vivo synthesized rice lectin

The synthesis and processing of rice lectin was followed in vivo in developing rice embryos. Using labelling and pulse-chase labelling experiments, the sequence of events in the synthesis and post-translational modifications of this protein could be determined. The primary lectin product observed in vivo is a high molecular weight precursor (28 K), which is post-translationally converted to a 23 K lectin protein, and in a further step cleaved into two smaller 12 K and 10 K polypeptides. The first step of the processing of the rice lectin is a rather slow process (the precursor has a half-life of about 3 h) and resembles the so-called vectorial processing of cytoplasmically made organellar proteins. The second modification consists of a (slow) proteolytic cleavage of the basic lectin subunit into two smaller polypeptides and resembles somewhat the cleavage of some legume (storage) proteins in their protein bodies.