Willy J. Peumans

Carbohydrate-binding Agents Cause Deletions of Highly Conserved Glycosylation Sites in HIV GP120 A NEW THERAPEUTIC CONCEPT TO HIT THE ACHILLES HEEL OF HIV

Mannose-binding proteins derived from several plants (i.e. Hippeastrum hybrid and Galanthus nivalis agglutinin) or prokaryotes (i.e. cyanovirin-N) inhibit human immunodeficiency virus (HIV) replication and select for drug-resistant viruses that show profound deletion of N-glycosylation sites in the GP120 envelope (Balzarini, J., Van Laethem, K., Hatse, S., Vermeire, K., De Clercq, E., Peumans, W., Van Damme, E., Vandamme, A.-M., Bolmstedt, A., and Schols, D. (2004) J. Virol. 78, 10617-10627; Balzarini, J., Van Laethem, K., Hatse, S., Froeyen, M., Van Damme, E., Bolmstedt, A., Peumans, W., De Clercq, E., and Schols, D. (2005) Mol. Pharmacol. 67, 1556-1565). Here we demonstrated that the N-acetylglucosamine-binding protein from Urtica dioica (UDA) prevents HIV entry and eventually selects for viruses in which conserved N-glycosylation sites in GP120 were deleted. In contrast to the mannose-binding proteins, which have a 50-100-fold decreased antiviral activity against the UDA-exposed mutant viruses, UDA has decreased anti-HIV activity to a very limited extent, even against those mutant virus strains that lack at least 9 of 22 (∼40%) glycosylation sites in their GP120 envelope. Therefore, UDA represents the prototype of a new conceptual class of carbohydrate-binding agents with an unusually specific and targeted drug resistance profile. It forces HIV to escape drug pressure by deleting the indispensable glycans on its GP120, thereby obligatorily exposing previously hidden immunogenic epitopes on its envelope.

Profile of resistance of human immunodeficiency virus to mannose-specific plant lectins.

ABSTRACT The mannose-specific plant lectins from the Amaryllidaceae family (e.g., Hippeastrum sp. hybrid and Galanthus nivalis) inhibit human immunodeficiency virus (HIV) infection of human lymphocytic cells in the higher nanogram per milliliter range and suppress syncytium formation between persistently HIV type 1 (HIV-1)-infected cells and uninfected CD4+ T cells. These lectins inhibit virus entry. When exposed to escalating concentrations of G. nivalis and Hippeastrum sp. hybrid agglutinin, a variety of HIV-1(IIIB) strains were isolated after 20 to 40 subcultivations which showed a decreased sensitivity to the plant lectins. Several amino acid changes in the envelope glycoprotein gp120, but not in gp41, of the mutant virus isolates were observed. The vast majority of the amino acid changes occurred at the N glycosylation sites and at the S or T residues that are part of the N glycosylation motif. The degree of resistance to the plant lectins was invariably correlated with an increasing number of mutated glycosylation sites in gp120. The nature of these mutations was entirely different from that of mutations that are known to appear in HIV-1 gp120 under the pressure of other viral entry inhibitors such as dextran sulfate, bicyclams (i.e., AMD3100), and chicoric acid, which also explains the lack of cross-resistance of plant lectin-resistant viruses to any other HIV inhibitor including T-20 and the blue-green algae (cyanobacteria)-derived mannose-specific cyanovirin. The plant lectins represent a well-defined class of anti-HIV (microbicidal) drugs with a novel HIV drug resistance profile different from those of other existing anti-HIV drugs.

Jasmonic acid methyl ester induces the synthesis of a cytoplasmic/nuclear chito-oligosaccharide binding lectin in tobacco leaves

In contrast to animal lectins, no evidence has indicated the occurrence of plant lectins, which recognize and bind “endogenous” receptors and accordingly are involved in recognition mechanisms within the organism itself. Here we show that the plant hormone jasmonic acid methyl ester (JAME) induces in leaves of Nicotiana tabacum (var. Samsun NN) the expression of a lectin that is absent from untreated plants. The lectin specifically binds to oligomers of N‐acetylglucosamine and is detected exclusively in the cytoplasm and the nucleus. Both the subcellular location and specificity indicate that the Nicotiana tabacum agglutinin (called Nictaba) may be involved in the regulation of gene expression in stressed plants through specific protein‐carbohydrate interactions with regulatory cytoplasmic/nuclear glycoproteins. Searches in the databases revealed that many flowering plants contain sequences encoding putative homologues of the tobacco lectin, which suggest that Nictaba is the prototype of a widespread or possibly ubiquitous family of lectins with a specific endogenous role.

Mutational Pathways, Resistance Profile, and Side Effects of Cyanovirin Relative to Human Immunodeficiency Virus Type 1 Strains with N-Glycan Deletions in Their gp120 Envelopes

ABSTRACT Limited data are available on the genotypic and phenotypic resistance profile of the α-(1-2)mannose oligomer-specific prokaryotic lectin cyanovirin (CV-N). Therefore, a more systematic investigation was carried out to obtain a better view of the interaction between CV-N and human immunodeficiency virus type 1 (HIV-1) gp120. When HIV-1-infected CEM cell cultures were exposed to CV-N in a dose-escalating manner, a total of eight different amino acid mutations exclusively located at N-glycosylation sites in the envelope surface gp120 were observed. Six of the eight mutations resulted in the deletion of high-mannose type N-glycans (i.e., at amino acid positions 230, 332, 339, 386, 392, and 448). Two mutations (i.e., at position 136 and 160) deleted a complex type N-glycan in the variable V1/V2 domain of gp120. The level of phenotypic resistance of the mutated virus strains against CV-N generally correlated with the number of glycan deletions in gp120, although deletion of the glycans at N-230, N-392, and N-448 generally afforded a more pronounced CV-N resistance than other N-glycan deletions. However, the extent of the decrease of antiviral activity of CV-N against the mutated virus strains was markedly less pronounced than observed for α(1-3)- and α(1-6)-mannose-specific plant lectins Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA), which points to the existence of a higher genetic barrier for CV-N. This is in agreement with a more consistent suppression of a wider variety of HIV-1 clades by CV-N than by HHA and GNA. Whereas the antiviral and in vitro antiproliferative activity of CV-N can be efficiently reversed by mannan, the pronounced mitogenic activity of CV-N on peripheral blood mononuclear cells was unaffected by mannan, indicating that some of the observed side effects of CV-N are unrelated to its carbohydrate specificity/activity.

The Abundant Class III Chitinase Homolog in Young Developing Banana Fruits Behaves as a Transient Vegetative Storage Protein and Most Probably Serves as an Important Supply of Amino Acids for the Synthesis of Ripening-Associated Proteins

Analyses of the protein content and composition revealed dramatic changes in gene expression during in situ banana (Musa spp.) fruit formation/ripening. The total banana protein content rapidly increases during the first 60 to 70 d, but remains constant for the rest of fruit formation/ripening. During the phase of rapid protein accumulation, an inactive homolog of class III chitinases accounts for up to 40% (w/v) of the total protein. Concomitant with the arrest of net protein accumulation, the chitinase-related protein (CRP) progressively decreases and several novel proteins appear in the electropherograms. Hence, CRP behaves as a fruit-specific vegetative storage protein that accumulates during early fruit formation and serves as a source of amino acids for the synthesis of ripening-associated proteins. Analyses of individual proteins revealed that a thaumatin-like protein, a β-1,3-glucanase, a class I chitinase, and a mannose-binding lectin are the most abundant ripening-associated proteins. Because during the ripening of prematurely harvested bananas, similar changes take place as in the in situ ripening bananas, CRP present in immature fruits is a sufficient source of amino acids for a quasi-normal synthesis of ripening-associated proteins. However, it is evident that the conversion of CRP in ripening-associated proteins takes place at an accelerated rate, especially when climacteric ripening is induced by ethylene. The present report also includes a discussion of the accumulation of the major banana allergens and the identification of suitable promoters for the production of vaccines in transgenic bananas.

Phylogenetic and specificity studies of two-domain GNA-related lectins: Generation of multispecificity through domain duplication and divergent evolution

A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes.

Plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle

n Abstractn n We describe the antiviral activity of plant lectins with specificity for different glycan structures against the severe acute respiratory syndrome coronavirus (SARS-CoV) and the feline infectious peritonitis virus (FIPV) in vitro. The SARS-CoV emerged in 2002 as an important cause of severe lower respiratory tract infection in humans, and FIPV infection causes a chronic and often fatal peritonitis in cats. A unique collection of 33 plant lectins with different specificities were evaluated. The plant lectins possessed marked antiviral properties against both coronaviruses with EC50 values in the lower microgram/ml range (middle nanomolar range), being non-toxic (CC50) at 50–100μg/ml. The strongest anti-coronavirus activity was found predominantly among the mannose-binding lectins. In addition, a number of galactose-, N-acetylgalactosamine-, glucose-, and N-acetylglucosamine-specific plant agglutinines exhibited anti-coronaviral activity. A significant correlation (with an r-value of 0.70) between the EC50 values of the 10 mannose-specific plant lectins effective against the two coronaviruses was found. In contrast, little correlation was seen between the activity of other types of lectins. Two targets of possible antiviral intervention were identified in the replication cycle of SARS-CoV. The first target is located early in the replication cycle, most probably viral attachment, and the second target is located at the end of the infectious virus cycle.n n

Two Distinct Jacalin-Related Lectins with a Different Specificity and Subcellular Location Are Major Vegetative Storage Proteins in the Bark of the Black Mulberry Tree

Using a combination of protein isolation/characterization and molecular cloning, we have demonstrated that the bark of the black mulberry tree (Morus nigra) accumulates large quantities of a galactose-specific (MornigaG) and a mannose (Man)-specific (MornigaM) jacalin-related lectin. MornigaG resembles jacalin with respect to its molecular structure, specificity, and co- and posttranslational processing indicating that it follows the secretory pathway and eventually accumulates in the vacuolar compartment. In contrast, MornigaM represents a novel type of highly active Man-specific jacalin-related lectin that is synthesized without signal peptide or other vacuolar targeting sequences, and accordingly, accumulates in the cytoplasm. The isolation and cloning, and immunocytochemical localization of MornigaG and MornigaM not only demonstrates that jacalin-related lectins act as vegetative storage proteins in bark, but also allows a detailed comparison of a vacuolar galactose-specific and a cytoplasmic Man-specific jacalin-related lectin from a single species. Moreover, the identification of MornigaM provides the first evidence, to our knowledge, that bark cells accumulate large quantities of a cytoplasmic storage protein. In addition, due to its high activity, abundance, and ease of preparation, MornigaM is of great potential value for practical applications as a tool and bioactive protein in biological and biomedical research.

The identification of inducible cytoplasmic/nuclear carbohydrate-binding proteins urges to develop novel concepts about the role of plant lectins.

During the last few years compelling evidence has been presented for the occurrence of cytoplasmic/nuclear plant lectins that are not detectable in normal plants but are only induced upon application of well-defined stress conditions. Since both the regulation of the expression and the subcellular location indicate that these ‘non-classical lectins’ are good candidates to play a physiologically important role as mediators of specific protein-carbohydrate-interactions within the plant cell, a critical assessment is made of the impact of these findings on the development of novel concepts about the role of plant lectins. Based on an analysis of the biochemical, molecular and evolutionary data of a jasmonate-induced chitin-binding lectin from tobacco leaves and a salt/jasmonate-induced leaf lectin from rice it is concluded that these lectins most probably interact with endogenous glycans located within the cytoplasmic/nuclear compartment of the plant cell. Several working mechanisms are proposed to explain how these inducible lectins may fulfill an important regulatory or structural role in stressed cells. In addition, the question of the evolutionary relationship(s) between the newly discovered inducible lectins and their ‘classical’ constitutively expressed homologs is addressed. Evidence is presented that the ‘non-classical lectins’ represent the main evolutionary line and that some of their corresponding genes were used as templates for genes encoding storage protein-like ‘classical’ homologs. Published in 2004.

Ectopically expressed leaf and bulb lectins from garlic (Allium sativum L.) protect transgenic tobacco plants against cotton leafworm (Spodoptera littoralis)

The insecticidal activity of the leaf (ASAL) and bulb (ASAII) agglutinins from Allium sativum L. (garlic) against the cotton leafworm, Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) was studied using transgenic tobacco plants expressing the lectins under the control of the constitutive CaMV35S promoter. PCR analysis confirmed that the garlic lectin genes were integrated into the plant genome. Western blots and semi-quantitative agglutination assays revealed lectin expression at various levels in the transgenic lines. Biochemical analyses indicated that the recombinant ASAL and ASAII are indistinguishable from the native garlic lectins. Insect bioassays using detached leaves from transgenic tobacco plants demonstrated that the ectopically expressed ASAL and ASAII significantly (Pxa0<xa00.05) reduced the weight gain of 4th instar larvae of S. littoralis. Further on, the lectins retarded the development of the larvae and their metamorphosis, and were detrimental to the pupal stage resulting in weight reduction and lethal abnormalities. Total mortality was scored with ASAL compared to 60% mortality with ASAII. These findings suggest that garlic lectins are suitable candidate insect resistance proteins for the control of S. littoralis through a transgenic approach.