Microbial lectin cofunction with lytic activities as a model for a general basic lectin role.

Lectins are ubiquitous proteins, which exhibit a specific and reversible sugar-binding activity. They react with glycosylated macromolecules and cells and may coaggragate them and lead to their lysis or alterations. Various lectin biological effects are well known, but their basic biological function is considered as yet unknown. In the present review, an experimental evidence and theoretical considerations are forwarded for supporting our suggestion that the general basic lectin or lectinoid (lectin-like protein) function in microorganisms, plants and animals is a cofunction enabling the activities of key lytic enzymes (lysins: glycosidases, proteases, esterases, phosphatases, hemolysin, etc.). The lectin service is: homing onto glycosylated receptors, anchoring to them and induction of cooperative conformational effects which enable their counterpart lysin activity on exogenous or endogenous target molecules and cells. The 'lectin-lysin' pair may reside in the same molecule, or in linked subunits. It may also be formed by cofunction of two separate entities originating from one or two (homogenous or heterogenous) cell sources. The lectin and lysin may be free or cell-bound components located intra or extracellularly. The final result of their cofunction is practically irreversible; either cell and macro-molecule lysis for nutrition, homeostasis and protection or cell alteration, reorganization and new productivity. Our suggestion emphasizes the prominent analogy of lectins to lytic enzyme positioning sites (LEPS), immunoglobulins and polypeptide hormones. The lectin analogy to LEPS and immunoglobulins is exhibited in the lectin-dependent cell and macromolecule lysis for nutritional and homeostatic purposes or for protection, respectively. The hormone-like lectin activity is exhibited in the lectin-dependent cell alterations. In addition to similar functions and effects, the analogy also includes the properties and behavior of these proteins. The suggested hypothesis is based on experimental evidence from microorganisms, plants and animals. It envisions the lectin and lectinoid function in cell attacks on glycosylated molecules or cells, cell-substratum and cell-cell interactions (fusion, invasion, etc.), cell transformation and formation of special structures. All of them according to a developmental program, or special (especially unfavourable) environmental conditions. The lectin resistance to proteolysis and unfavourable pH or temperature is in accord with the suggested hypothesis.


SUMMARY
Lectins are ubiquitous proteins, which exhibit a specific and reversible sugar-binding activity. They react with glycosylated macromolecules and cells and may coaggregate them and lead to their lysis or alterations. Various lectin biological effects are well known, but their basic biological function is considered as yet unknown. In the present review, an experimental evidence and theoretical considerations are forwarded for supporting our suggestion that the general basic lectin or lectinoid (lectin-like protein) function in microorganisms, plants and animals is a cofunction enabling the activities of key lytic enzymes (lysins: glycosidases, proteases, esterases, phosphatases, hemolysin, etc.). The lectin service is: homing onto glycosylated receptors, anchoring to them and induction of cooperative conformational effects which enable their counterpart lysin activity on exogenous or endogenous target molecules and cells. The 'lectin-lysin' pair may reside in the same molecule, or in linked subunits. It may also be formed by cofunction of two separate entities originating from one or two (homogenous or heterogenous) cell sources. The lectin and lysin may be free or cell-bound components located intra or extracellularly. The final result of their cofunction is practically irreversible; either cell and macromolecule lysis for nutrition, homeostasis and protection or cell alteration, reorganization and new productivity.
Our suggestion emphasizes the prominent analogy of lectins to lytic enzyme positioning sites (LEPS), immunoglobulins and polypeptide hormones. The lectin analogy to LEPS and immunoglobuhns is exhibited in the lectin-dependent cell and macromolecule lysis for nutritional and homeostatic purposes or for protection, respectively. The hormone-like lectin activity is exhibited in the lectin-dependent cell alterations. In addition to similar functions and effects, the analogy also includes the properties and behavior of these proteins. The suggested hypothesis is based on experimental evidence from microorganisms, plants and animals. It envisions the lectin and lectinoid function in cell attacks on glycosylated molecules or cells, cell-substratum and cell-cell interactions (fusion, invasion, etc.), cell transformation and formation of special structures. All of them according to a developmental program, or special (especially unfavourable) environmental 0168-6445/89/$03.50 © 1989 Federation of European Microbiological Societies conditions. The lectin resistance to proteolysis and unfavourable pH or temperature is in accord with the suggested hypothesis.

INTRODUCTION
The first description of lectin hemagglutinating activity, by the protein ricin of Ricinus communis seeds, was forwarded by Stillmark 100 years ago [1]. This hemagglutination was shown to be reversible and inhibitable by D-galactose. During the following century, many additional lectins, specifically inhibitable by various sugars, were described in plants, animals and microorganisms [2][3][4][5][6]. Their specific binding was compared to that of enzymes and antibodies [4], which were both excluded from lectins by definition [7].
The lectins, including those of microorganisms, have been shown to be very useful for various scientific, medical, agricultural and biotechnological systems, including [3,8]: detection of sugars in solutions, on macromolecules and cells, detection and identification of blood groups and bacteria, protein purification and cell fractionation, mitogenic stimulation of peripheral lymphocytes [9], examination of chromosomes and diagnosis of genetic or chronic diseases characterized by immunodeficiency (cancer, etc.), as well as reduction of cancer cell tumorigenicity [8,10]. They have been demonstrated to be involved in various biological systems, including: adhesion of symbiotic [11] and pathogenic [12] microbes to their host for the establishment of infection, and the adhesion of cancer cells to tissues and organs for the production of metastases [13][14][15][16][17]. They have also been described as being involved in protecting eukaryotic organisms against infecting microorganisms [18], by enabling their lysis [19] or phagocytosis [20,21], and in the removal of senescent macromolecules and cells [22][23][24] by similar mechanisms. However, the central question in lectinology --the general or basic lectin function was not answered. Based on our studies on Pseudomonas aeruginosa, and the relevant literature on microbial, plant and animal lectins, we offer the suggestion that the general basic lectin role is a cofunction with lytic entities (lysins) analogous to  (1) and on cell surface, bridged by its membrane (2). * B which is schematically represented as one binding site is mostly multimeric.
that of lytic enzyme 'positioning sites' (LEPS) [25], immunoglobulins and hormones. The lectin lysin pair may exist on the same or adjacent molecules ( Fig. 1), originating from the same cell or from different cellular sources. Their cooperative function may lead to either cell and macromolecule lysis (LEPS and antibody-like lectin activity) or to cell alteration, reorganization and production of new structures or products (hormone-like lectin activity). The support for this hypothesis is based on a wide experimental evidence, indicating: (a) a close structural, genetic and physiological association between lectin and lysin production (paragraph 3) (b) a striking analogy between lectin, LEPS and immunoglobulin function, properties and behavior (paragraph 4), (c) numerous examples for lectin cofunction with coexisting lytic activities (paragraph 5), (d) a clear analogy between lectin and polypeptide hormone function and effects (paragraph 6), as well as (e) a broad experimental evidence showing that inhibition of the reversible lectin binding by sugars, prevents the irreversible effects induced by the associated lysins (paragraph 7).

AN EVIDENCE FOR A CLOSE STRUCT-URAL, GENETIC AND PHYSIOLOGICAL ASSOCIATION BETWEEN LECTINS AND LYSIN PRODUCTION
The evidence for genetic-physiological association between lectins and lysins includes: (a) the lectinoid enzymes and toxins which are composed of both components in the same molecule, or A--exhibiting a lytic or toxic activity, and B--a lectin or lectinoid (lectin-like protein, confronting with the classical lectin definition [7] due to lack of either coaggregating activity or defined sugar specificity). The toxic A domains of some bacterial exotoxins (e.g. Corynebacterium diphtheriae and Pseudomonas aeruginosa) block protein synthesis in the attacked target eukaryotic cells by ADPribosylation of elongation factor-2 (EF-2) in the presence of NAD [26,27]. Similarly, A domains of some plant [28][29][30][31][32][33] and shiga [34] toxins inhibit protein synthesis via ribosomal deterioration mediated by N-glycosidase [29][30][31][32]. Other powerful bacterial toxins (cholera, tetanus, botulinus, etc.) also exhibit a similar A-B structure and cofunction. Removal of B from A, does not necessarily prevent the latter in vitro lytic activity on its 213 isolated cell-free substrate, but prevents its effect on the intact cells ( Fig. 2:3) and the intracellularly located substrate [26][27][28][29][30][31][32]. During our studies on Pseudornonas aeruginosa lectins, we have found that there is a physiological and genetical association between lectin production and protease, hemolysin and pyocyanin levels (8,(35)(36)(37). Lectin-lacking strains were devoid of the lytic activities and lectin-deficient strains and mutants had low levels of these activities. Reciprocal confirmation of the linkage was attained by the demonstration that genetically defined protease-deficient mutants of P. aeruginosa were found to be poor lectin producers [38]. The resistance of P. aeruginosa lectins to protease (a property also shared by many plant and animal lectins) fits the cofunction hypothesis. A genetic association between lectinoid adhesins and hemolysin may also be deduced from the literature describing the cloning of the adhesin-encoding genes in Escherichia coli [39,40] and from the simultaneous existence of a sialophilic lectin and sialidase in the membrane of influenza A virus [41][42][43]. Cell surface-bound A and B domains, either linked in the same molecule, or bridged by the cell membrane, may function like the A-B-containing cellfree macromolecules (Fig. 1).
Examples from plants and animals include the a-galactosidases with lectin activities in Vicia faba [44], the temporal regulation of the production of the lectin Con A and a-mannosidase in the seeds of Canavalia ensiformis [45,46] and the lectin-linked sialyltransferase on Hodgkin's cells [47]. Additional examples of lectin-lysin coexistence in microorganisms may be deduced from the literature describing tectin or hemagglutinin purification from associated lysins [2].

THE ANALOGY BETWEEN LECTINS, LEPS AND ANTIBODIES
In addition to their lytic active site the lytic enzymes, acetylcholinesterase and chymotrypsin, display a 'positioning site' [25], LEPS, which is involved in their homing and binding to the substrate 'positioning group' (Fig. 3:1,2). This binding enables best exposure of the substrate sensitive group to the enzyme lytic site for its decomposition [25]. In a similar way antibody binding to antigen-bearing macromolecules or cells conditions their lysis by complement ( Fig. 3:4). We suggest that lectin role ( Fig. 3:3) is analogous to that of the above described LEPS and antibodies. They behave as 'saccharophilic LEPS' or antibodies cofunctioning with various lysins. The lectin or lectinoid specificity for glycosylated molecules ensures their homing to cells and to extracellular matrix (glycocalyx), which are specified by glycosylated receptors. They bind to sensitive cells (displaying the compatible sugars on their surface) and induce membranal changes which enable the entrance of the A domain into the cell for attaining contact with its sensitive intracellular target [26][27][28][29][30][31][32][33]. The lysins, which may reside with the lectins in the same molecule or in adjacent location, display various lytic or toxic activities, inchiding: sialidase [41][42][43]. O-glycosidases [44], Nglycosidases [29][30][31][32], glycosyl or sialyl transferases [47], NAD-ADP ribosyltransferase of EF-2 [26,27], proteases [48], and hemolytic activities. If the purpose of the lysis is nutritional, it may be compared to chymotrypsin activity, if it is homeostatic, to acetylcholinesterase and if protective, to the immune lysis.
In addition to the above discussed lysis-enabling receptor-binding cofunction, the lectins share with LEPS and immunoglobulins several other features, including: (1) A wide distribution in nature. Lectins and LEPS are ubiquitous and immunoglobulins are produced in vertebrates.
(2) Multimeric structure and cooperative interactions, which may exhibit allosteric regulation and induce conformational-structural alterations in cell membranes and mactomolecules.
In the absence of inactivation of the associated A lytic counterpart the irreversible lytic process is prevented, but B still binds to the target molecules and cells (Fig. 2:3) and may coaggregate them (pricipitate macromolecules and agglutinate cells).
The binding and aggregation are sensitive to competitive inhibition by sugars. (8) Resistance to the lytic action of their counterparts and to the essentially unfavourable conditions inducing their production and function. (9) Their production is constitutively low and inducibly massive, increasing only when needed [49][50][51] and fading after the termination of their function. (10) Cells and macromolecules which lack specific receptors for them (the positioning groups, specific sugars and antigens, respectively), as an essential trait, or due to receptor removal by enzymes, are resistant to the reversible coaggregating and the irreversible lytic effects (Fig. 2 : 4).
A most interesting and profound support for the above described analogy between lectins, LEPS and antibodies, is their overlaping functions and mutual replacement. Examples for LEPS replace- ment by lectins are the lectinoid glycosidases [44] protease [48] and glycosyl or ADP-ribosyl transferases [26,27]. Antibody-like lectin activity is well known in invertebrates [4,18,19,51,52] and other organisms devoid of immunoglobulins, including the vertebrate embryo [51], as well as in vertebrate non-immune phagocytosis [20,21] (Figs. 5 and 6). The lectin specificity is lower (broader reactivity) than that of antibodies. Lectin-mediated trapping and degradation of sugar-bearing macromolecules or cells (Figs. 5-7) was also described in hepatocytes [22] and in other cell types, including free living unicellulars [5]. Similarly, most cells and many macromolecules bear sugars which react with such lectins.
In addition to the lectinoid enzymes, there are numerous examples of subcellular microbial and plant lectin or lectinoid cohesion (not via the sugar-binding site) with lysins and toxins [2]. These include descriptions of purified lectin contamination by lytic factors [2] and reports on pure lectin effects (e.g. inhibition of fungus growth by the purified chitin-binding lectin of wheat germ ag-glutinin [53]), which were later found to be due to contamination by lyric activities (e.g. chitinase activity). A considerable direct evidence for lectin interaction with hydrolytic enzymes is found in the literature describing the interaction of immobilized lectins (lecrins bound to Sepharose columns) with such enzymes. The interaction between Con A and the a-mannosidase and agalactosidase [54] of Canavalia ensiformis seeds [45,46] is a well known example.
A clear cut example of cell surface lectin cofunction with adjacent lyric activity ( Fig. 1 : 2) is that the influenza A virus sialophilic lectin and sialidase [41][42][43]. The lectin homing onto sialic acid-bearing cells facilitates the degradation of the cells receptors by the sialidase and enables the following membranal fusion [55,56]. Surface lectins and lectinoid adhesins of many other microorganisms also condition their lysin attacks on the host cells (Fig. 7) [6,8,57,58].
Among the fungal examples are the insect-attacking Conidiobolus lamprauges, which produces a chitin-binding lectin and accompanying chitindegrading activity, hemolysin and protease [57] and the lectins of the nematophagous fungi [58]. Animal examples include the phagocyte and hepatocyte membranal lectins which enable the degradation of target molecules by the intracellu- 217 lar lysosomal enzymes of these cells (Fig. 8 : top). The hepatocyte membranal lectin is involved in the removal of circulating desialylated glycoproteins [21] leading to their decomposition by the intracellular lysosomal enzymes. Internal hepatocyte lectins were reported to contribute to the lysosomal enzyme trafficking [59]. The cofunction may also involve lectin and lyric entities of heterologous origins (Figs. 6:PA and 8:middle and bottom). An example is the microbial lectins or lectinoids which are involved in trapping (and subsequent lysis) of the bacteria which bear them [20,21] and other bacteria [20] (Fig. 6) by phagocytes. Another example is animal cell lysis by bacterial or cancer cells due to cofuncrion of the target cell lectins with the lysins of the attacking cells. The condition is similar to that described in Fig. 7, but instead of the bacterial lectin binding to the host cell sugars, the host cell lectins interact with the microbial sugars, leading to the same result.

THE ANALOGY BETWEEN LECTINS AND POLYPEPTIDE HORMONES
Exogenous lectin interaction with cell membrane receptors (Fig. 8:middle), or endogenous lectin interaction with exogenous glycosylated macromolecules, may enable endogenous key lytic activities which trigger a cascade of cellular reactions culminating in hormone-like induced cell transformation. A well known example is the insulin-like effects of Con A and wheat germ agglutinin [60]. The involvement of key lyric enzymes in this effect may be supported by the facts that: (a) both hormones and lectins were reported to enable phosphoinositide hydrolysis and subsequent activation of protein kinase [61] and (b) trypsin activity was reported to similarly affect the system [62]. The mitogenic stimulation of peripheral lymphocytes [9] is an example for hormonelike lectin activity. These hormone-like lectin-dependent phenomena may also be specifically inhibited by adding sugar in the initial stage of the lectin reversible interaction with the cell surface before enabling the triggering lyric activities. Polypeptide hormones also share (with lectins, LEPS and immunoglobulins) most of the properties described in paragraph 4.

EXPERIMENTAL EVIDENCE SHOWING THAT INHIBITION OF THE LECTIN OR LECTINOID REVERSIBLE INTERACTION COMPETITIVELY INHIBITS THE ASSOCI-ATED IRREVERSIBLE LYTIC PROCESSES
The practically irreversible hydrolytic decomposition of acetylcholine by the esteratic site of acetylcholinesterase is competitively inhibited by compounds which bind to the LEPS of this enzyme ( Fig. 4 : 1). Similarly, the irreversible lysis of antigen-beating macromolecules and cells by complement is competitively inhibited by haptens which bind to the antibody ( Fig. 4 : 3). The same rule exists also in the lectin-dependent lyric system: sugars may competitively inhibit the lyric activities associated with the lectins (Figs. 2 : 2 and 4:2). This rule is an important component in the theoretical considerations of the herein suggested lectin-lysin cofunction hypothesis. Therefore, we forward a wide-spectrum experimental evidence from microorganisms (viruses, bacteria and fungi), plants and animals, showing that prevention of irreversible lytic activities is obtained by an early competitive, or non-competitive, inhibition of the reversible lectin (or lectinoid) binding to their receptors. The competitive inhibition may be obtained by sugars (Figs. 2:2 and 4:2) or by competing foreign lectins. The non competitive inhibition may be based on inactivation of the lectin (or lectinoid), as well as on masking or removal of its target cell receptors (Fig. 2). These are represented by the following examples: (1) Addition of L-rhamnose (6-deoxy-L-mannose), or a lectin from Streptomyces which binds this sugar, competitively inhibits the lysis of the bacterium Lactobacillus casei by its bacteriophage [63]. Similarly, Con A competitively inhibits lysis of Bacillus subtilis by its bacteriophage [64,65].
(2) The fusion of influenza A virus with eukaryotic cell membrane, culminating in lysis of the latter, was shown to be dependent on its lectin [41][42][43]55,56]. Desialylated cells are resistant to the virus.
(3) The lyric activity of nematophagous fungi is inhibited by sugars [58]. (4) The toxic and cytolytic effects of the lectinoid microbial (cholera, shiga, diphtheria, Pseudornonas, tetanus and botulinus) toxins are inhibited by sugars or glycoconjugates resembling the glycosylated membrane lipid or protein receptors of the sensitive cells [26,27]. The interesting point in this regard is the fact that Bacillus thuringiensis glycosylated toxin insecticidal activity, caused by its interaction with lectinoid receptors in the larval mosquito gut, is also inhibited by sugars [66]. (5) The plant toxins (ricin, abrin, modeccin, isidor, etc.) are similarly inhibited by sugars [28][29][30] (6) The lectin-dependent microbial lysis in vertebrates and invertebrates was shown to be blocked by sugars [19][20][21] or by competing lectins [20]. (7) The degradation of circulating desialylated glycoproteins by hepatocytes is inhibited by removal of their unmasked galactose residues which are trapped by the hepatocyte surface lectins [22]. (8) Cancer cell metastases are prevented by sugars which bind to the cancer cell [13,14,17] or to the liver lectins [15,16], depending on which one of them enables the cancerous cell settlement.
The lectin or lectinoid (either exogenous or endogenous) importance for the targeting of the lysin (bound to either lectin or glycosylated molecules) to sensitive cells has found an important application in cancer lectinotherapy [51,[67][68][69][70]. Lectins or lectinoids (as well as monoclonal antibodies) which recognize the cancer cell receptors, or glycoconjugates which are recognized by the cancer cell lectins (or lectinoids) may be used for targeting drugs (or monoclonal antibodies) to these cells.

CONCLUSIONS AND FUTURE WORK
According to the herein suggested hypothesis, lectin basic role is that of a cofactor which enables the function of key lytic activities. The latter lead to either cell and macromolecule lysis for nutritional, homeostatic and protective purposes or trigger cell alteration and reorganization needed for developmental, functional or survival pur-poses. Future work is desired for confirmation of this suggestion, which envisions lectins as lysisenabling receptor-binding cofactors, including: (1) Looking for the lytic counterparts of known lectins. Many of them are yet unknown. Their discovery and the study of their role may contribute enormously to the understanding of lectin functions.
(2) Looking for new lectin-lytic pairs in systems involving profound macromolecule or cell (including cell population) lysis or transformations (developmental; malignant, or environmentally induced): cell growth or division, sporulation, fertilization, germination, cell-substratum and cell-cell interactions (symbiosis, commensalism or parasitism, cell protection, fusion, and wound healing). Lectin interaction with exogenous glycosylated macromolecules or cells may also lead to conformational changes which enable the adjacent lysin activity. In many of these conditions appearance and involvement of lectins [49,50] and special metabolic activities were separately described, mostly without demonstration of a lysin-lectin linkage.
(3) Construction of neo lectin-lysin pairs (or half pairs which would cooperate with existing counterparts in the target cells) and examination of their biological effects and applications for medical, industrial, biotechnological and other scientific and applicative fields. (4) Examination of lectin involvement in lytic phenomena and cell alterations which are induced by glycocosylated macromolecules.