Metabolic labeling of sialic acids in tissue culture cell lines: methods to identify substituted and modified radioactive neuraminic acids

The parent sialic acid N-acetylneuraminic acid can be modified or substituted in various ways, giving rise to a family of more than 25 compounds. The definitive identification of these compounds has previously required isolation of nanomole amounts for mass spectrometry or NMR. We have explored the possibility of using the known metabolic precursors of the sialic acids, particularly N-acetyl-[6-3H]mannosamine, to label and identify various forms of sialic acids in tissue culture cells. Firstly, we defined several variables that affect the labeling of sialic acids with N-acetyl-[6-3H]mannosamine. Secondly, we have devised a simple screening method to identify cell lines that synthesize substituted or modified sialic acids. We next demonstrate that it is possible to definitively identify the natures of the various labeled sialic acids without the use of mass spectrometry, even though they are present only in tracer amounts. The methods used include paper chromatography, analytical de-O-acetylation, periodate release of the 9-3H as [3H]formaldehyde (which is subsequently converted to a specific 3H-labeled chromophore), acylneuraminate pyruvate lyase treatment with identification of [3H]acylmannosamines, gas-liquid chromatography with radioactive detection, and two new high-pressure liquid chromatography methods utilizing the amine-adsorption:ion suppression and ion-pair principles. The use of an internal N-acetyl-[4-14C]neuraminic acid standard in each of these methods assures precision and accuracy. The combined use of these methods now allows the identification of radioactive tracer amounts of the various types of sialic acids in well-defined populations of tissue culture cells; it may also allow the identification of hitherto unknown forms of sialic acids.     

Diversity of sialic acids revealed using gas chromatography/mass spectrometry of heptafluorobutyrate derivatives

The fine structural motifs of sialic acids, a frequent terminal monosaccharide of glycans, seem to contain essential biological properties. To identify such subtle structural differences, a reliable method was developed for the qualitative and quantitative identification of sialic acids present in different tissues and fluids. This method involved, after liberation of sialic acids by mild acid hydrolysis, their methyl esterification using diazomethane in the presence of methanol and the formation of volatile derivatives using heptafluorobutyric anhydride. The derivatives were analyzed by gas chromatography coupled to mass spectrometry in the electron impact mode. This technique allowed the separation and identification of a large variety of sialic acids, including different O-acylated forms of N-acetyl and N-glycolyl neuraminic acids and of 3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn). This method allowed also identifying 8-O-methylated and 8-O-sulfated derivatives, de-N-acetylated neuraminic acid, and 1,7-sialic acid lactones. Compounds present in very complex mixtures could be identified through their fragmentation patterns. Because of the stability of the heptafluorobutyrate derivatives, this method presents important improvements compared to the previous techniques, because it can be frequently applied on very small amounts of crude samples. This methodology will support progress in the field of the biology of sialic acids.     

Identification of 9-O-acetyl-N-acetylneuraminic acid in normal canine pre-ocular tear film secreted mucins and its depletion in Keratoconjunctivitis sicca

O-Acetylated sialic acids have been reported in many sialoglycoproteins where they mediate a variety of immune and other biological events. We have previously demonstrated that the protective mucus barrier on the surface of the canine eye contains sialoglycoproteins. We have also investigated the occurrence of O-Acetylated sialic acids in these ocular mucins. Mucus aspirated from the surface of normal dog eyes and those with keratoconjunctivitis sicca (KCS) was fractionated into three pools by density gradient centrifugation. Sialic acids comprised 0.6–0.9% of the dry weight of the mucins isolated. The sialic acid profile in these pools was examined using HPLC. O-Acetylated sialic acids, mainly Neu5,9Ac₂, were detected in normal animals and made up 10–30% of the total sialic acids detected. A doubling of the sialic acid content was found in KCS mucins, but the level of 9-O-Acetylated sialic acid was reduced below 4% of total. Histological analysis of conjunctival tissue from normal and KCS dogs showed the presence of sialic acids, detected with the α(2–6) sialic acid-specific lectin Sambucus nigra, in the goblet cells and corresponding to the staining pattern for MUC5AC, the major ocular-secreted mucin gene product. In KCS animals a disruption of the normal pattern of conjunctival goblet cells was seen with preservation of the pattern of lectin binding observed in normal animals. Thus the data demonstrate the presence of mono-O-Acetylated sialic acids in normal canine ocular mucins and a loss of this population of sialic acids in dry eye disease in spite of a significant increase in total sialic acids in KCS mucin.     

An ion-exchange chromatography procedure for the isolation and concentration of basic amino acids and polyamines from complex biological samples prior to high-performance liquid chromatography.

The original objective of this study was to develop a selective and sensitive method for the analysis and quantification of basic amino acids from biological samples via reversed-phase high-performance liquid chromatography. Using various previously described techniques for the separation of amino acids, we were unsuccessful in measuring levels of histidine, arginine, ornithine, and lysine in biological samples due to the presence of interfering compounds. A "cleanup" procedure for the isolation of the basic amino acids using a weakly acidic cation exchange resin, Biorex-70 (Bio-Rad), is described in detail. Upon separation from the bulk of the neutral and acidic amino acids, the basic amino acids were subjected to precolumn fluorescence derivatization using 9-fluorenylmethyl chloroformate (FMOC) and the fluorescent derivatives were separated by RP-HPLC. The advantages of this method over previously described amino acid analysis techniques are (i) isolation and stable recovery (greater than 95%) of the desired basic amino acids, (ii) sensitivity of detection (low pmol range), (iii) complete resolution of derivatized amino acids via HPLC, (iv) limited amount of sample required for analysis, and (v) samples readily concentrated by lyophilization or rotoevaporating. This ion-exchange cleanup procedure was also adapted for the analysis of polyamines in concentrated culture media samples and proved additionally advantageous by eliminating the use of costly C-18 extraction columns required by previously described techniques.     

A second uniquely human mutation affecting sialic acid biology

Siglecs are immunoglobulin superfamily member lectins that selectively recognize different types and linkages of sialic acids, which are major components of cell surface and secreted glycoconjugates. We report here a human Siglec-like molecule (Siglec-L1) that lacks a conserved arginine residue known to be essential for optimal sialic acid recognition by previously known Siglecs. Loss of the arginine from an ancestral molecule was caused by a single nucleotide substitution that occurred after the common ancestor of humans with the great apes but before the origin of modern humans. The chimpanzee Siglec-L1 ortholog remains fully functional and preferentially recognizes N-glycolylneuraminic acid, which is a common sialic acid in great apes and other mammals. Reintroducing the ancestral arginine into the human molecule regenerates the same properties. Thus, the single base pair mutation that replaced the arginine on human Siglec-L1 is likely to be evolutionarily related to the previously reported loss of N-glycolylneuraminic acid expression in the human lineage. Siglec-L1 and its chimpanzee Siglec ortholog also have a different expression pattern from previously reported Siglecs because they are found on the lumenal edge of epithelial cell surfaces. Notably, the human genome contains several Siglec-like pseudogenes that have independent mutations that would have replaced the arginine residue required for optimal sialic acid recognition. Thus, additional changes in the biology of sialic acids may have taken place during human evolution.     

O-acetylation and de-O-acetylation of sialic acids. Sialic acid esterases of diverse evolutionary origins have serine active sites and essential arginine residues

We and others have recently described 9-O-acetyl-sialic acid esterase (9-O-Ac-SA esterase) activities that appear to be specific for removal of O-acetyl esters from the 9-position of naturally occurring sialic acids. We have now examined a variety of species for such enzymes and found them in vertebrates and higher invertebrates, but not in plants or in lower invertebrates. This evolutionary distribution correlates well with that of the sialic acids themselves. All of the 9-O-Ac-SA esterase activities tested were inhibited by diisopropyl fluorophosphate (DFP) in a dose-dependent fashion. This indicates that each of these enzymes has a serine active site similar to the well known serine esterases and serine proteases. Methyl esterification of the carboxyl group of 9-O-acetyl-N-acetylneuraminic acid significantly reduced the activity of all of the 9-O-Ac-SA esterases against the O-acetyl group. This indicates that each of these enzymes may recognize the negatively charged carboxyl group of the sialic acid. Enzymes that recognize anionic substrates frequently have an essential arginine residue (Riordan, J. F., McElvany, K. D., and Borders, C. L., Jr. (1977) Science 195, 884-886). We therefore studied the effects of the arginine-specific modifying reagents 2,3-butanedione and phenylglyoxal on 9-O-Ac-SA esterase activities from influenza C virus, human erythrocytes, rat liver, starfish gonads, and sea bass brain. All of these enzymes were inhibited in a dose-dependent fashion by both reagents, under conditions previously known to avoid nonspecific modification. In contrast, the typical serine proteases trypsin and kallikrein and the serine esterase acetylcholinesterase were not significantly affected, even by the highest concentrations of these reagents used. These data indicate that five 9-O-Ac-SA esterase activities from evolutionarily distinct origins all have serine active sites and essential arginine residues. We postulate that the arginine residue is involved in substrate recognition via the negatively charged carboxyl group of the sialic acids. Thus, these 9-O-Ac-SA esterase activities may be members of a previously undescribed class of serine esterase.     

Evaluation of high-performance anion-exchange chromatography with pulsed electrochemical and fluorometric detection for extensive application to the analysisof homologous series of oligo- and polysialic acids in bioactive molecules.

Our previous studies have shown extensively diverse structures in oligo/polymers of sialic acid (oligo/polySia) that are expressed often in developmentally regulated manner on animal glycoconjugates. The aim of this study was to establish highlysensitive and specific methods that can be used to identify diverse types of oligo/polySia and thus can be applied to studies of biological phenomena associated with the differential expression of oligo/polySia chains with different degree of polymerization (DP). As model compounds, we analyzed five different homologous series of oligo/polySia, (-->8Neu5Acalpha2-->)(n), (-->9Neu 5Acalpha2-->)(n), (-->8Neu5Gcalpha2-->)(n), (-->5-O(glycolyl)-Neu5Gcalpha2-->)(n), and Neu5Gc9SO(4)alpha2-->(-->5-O(glycolyl)-Neu5Gcalpha2--> )(n), ()expressed in various biopolymers. The latter two structures have recently been identified in sea urchin egg receptor for sperm. First we examined application of high-performance anion-exchange chromatography (HPAEC) on a CarboPac PA-100 column with pulsed electrochemical detection (PED) to new types of oligo/polySiacompounds and confirmed that resolution of high polymers (DP >70) of sialic acids was remarkable as reported previously. However, there are limitations in sensitivity and selectivity in PED that become significant when material is available only in a minute amount or material contained a large proportion of protein. These limitations can be circumvented by fluorometric detection of oligo/polySia tagged with 1,2-diamino-4, 5-methyl-enedioxybenzene (DMB) at the reducing terminal residues after separation on a MonoQ HR5/5 column. The latter method can be applied to any type of oligo/polySia we examined if we choose the derivatization conditions and is more sensitive and specific than the method with PED for analysis of oligo/polySia with DP up to 25.     

Derived structure of the putative sialic acid transporter from Escherichia coli predicts a novel sugar permease domain.

Catabolism of sialic acids by Escherichia coli requires the genes nanA and nanT, which were previously mapped between argG and rpoN (E.R. Vimr and F.A. Troy, J. Bacteriol. 164:845-853, 1985). This organization is confirmed and extended by physical mapping techniques. An open reading frame beginning 135 bp from the nanA translational stop codon could code for a 53,547-Da hydrophobic polypeptide predicted to contain 14 transmembrane segments. Complementation analysis confirmed that nanT is required for sialic acid uptake when expressed in trans. NanT is homologous to a putative permease encoded by open reading frame 425, which maps between leuX and fecE in the E. coli chromosome. However, unlike this hypothetical permease or previously reported monosaccharide transporters, NanT contains a centrally located domain with two additional potential membrane-spanning segments plus one amphiphilic alpha-helix that may be important for the structure and function of sialic acid-permease.     

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.     

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.     

Reversed-phase high-performance liquid chromatography separation of dimethylaminoazobenzene sulfonyl- and dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the picomole level

A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of cysteic acid, S-sulfocysteine, hydroxyproline, taurine, norleucine, cystine, and delta-hydroxylysine. The precolumn derivatization of amino acids with dimethylaminoazobenzene sulfonyl chloride (DABS-Cl) is simple and quick (10 min at 70 degrees C) and allows the complete reaction of primary and secondary amino acids. The separation of the compounds under investigation is achieved in 25 min using a reversed-phase 3-microns Supelcosil LC-18 column at room temperature. The versatility of the proposed method is documented by amino acid determination on protein samples obtained using different hydrolysis techniques (HCl, methane-sulfonic acid, and NaOH), with attention given to the detection of tryptophan in protein samples with high sugar concentration. Furthermore, we have reported the experimental conditions necessary to apply this method to the amino acid analysis of very low amount of proteins (1 to 5 micrograms) electroeluted from a stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stability of DABS-derivatives, the short time of analysis, the high reproducibility and sensitivity of the system, and the complete resolution of all compounds of interest make this method suitable for routine analysis. Furthermore, we have also developed a fast reversed-phase high-performance liquid chromatographic method for the complete separation of dimethylaminoazobenzene thiohydantoin (DABTH)-amino acids. The separation of the compounds under investigation is obtained, at room temperature, in less than 18 min using a reversed-phase Supelcosil LC-18 DB column, 3-micron particles, and also allows the complete separation of DABTH-Ile, DABTH-Leu, and DABTH-Norleu. The short time of analysis, together with the high reproducibility of the system and its sensitivity at picomole levels, make this method very suitable for the identification of DABTH-amino acids released during microsequencing studies of proteins and peptides with the dimethylaminoazobenzene isothiocyanate reagent. In addition, we have shown that it is possible to obtain complete separation of DABTH-amino acids also under isocratic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serum sialylation changes in cancer

Cancer is a major cause of death in both developing and developed countries. Early detection and efficient therapy can greatly enhance survival. Aberrant glycosylation has been recognized to be one of the hallmarks of cancer as glycans participate in many cancer-associated events. Cancer-associated glycosylation changes often involve sialic acids which play important roles in cell-cell interaction, recognition and immunological response. This review aims at giving a comprehensive overview of the literature on changes of sialylation in serum of cancer patients. Furthermore, the methods available to measure serum and plasma sialic acids as well as possible underlying biochemical mechanisms involved in the serum sialylation changes are surveyed. In general, total serum sialylation levels appear to be increased with various malignancies and show a potential for clinical applications, especially for disease monitoring and prognosis. In addition to overall sialic acid levels and the amount of sialic acid per total protein, glycoprofiling of specific cancer-associated glycoproteins, acute phase proteins and immunoglobulins in serum as well as the measurements of sialylation-related enzymes such as sialidases and sialyltransferases have been reported for early detection of cancer, assessing cancer progression and improving prognosis of cancer patients. Moreover, sialic-acid containing glycan antigens such as CA19–9, sialyl Lewis X and sialyl Tn on serum proteins have also displayed their value in cancer diagnosis and management whereby increased levels of these factors positively correlated with metastasis or poor prognosis.     

Separation methods for sialic acids and critical evaluation of their biologic relevance

Sialic acids are biosynthesized by almost all organisms as a 9-carbon carboxylated monosaccharide and are integral components of glycoconjugates. More than 40 naturally occurring sialic acid derivatives of the three main forms of sialic acids, the N-acetyl- and N-glycolylneuraminic acid and 2-keto-3-deoxy-nonulosonic acid have been identified. Due to the great importance of sialic acids as key mediators in a plethora of cellular events, including cell-cell recognition and cell-matrix interactions, their analysis in biologic samples is useful for a deeper understanding of the various (patho)physiological processes and of value in disease diagnosis and monitoring. In this review we summarize the methodology developed to isolate and liberate sialic acids from biologic samples as well as the chromatographic, electromigration and hyphenated techniques available for their separation and analysis. A critical evaluation of the biological relevance of the results obtained by analyzing sialic acids in biologic samples is also presented.     

Separation methods for camptothecin and related compounds

This paper reviews working procedures for the analytical determination of camptothecin and analogues. We give an overview of aspects such as the chemistry, structure–activity relationships, stability and mechanism of action of these antitumor compounds. The main body of the review describes separation techniques. Sample treatment and factors influencing high-performance liquid chromatography development are delineated. Published high-performance liquid chromatographic methods are summarized to demonstrate the variability and versatility of separation techniques and a critical evaluation of separation efficiency, detection sensitivity and specificity of these methods is reported.     

Characterization of mono-, di-, and tri-O-acetylated sialic acids on human cells

The presence of mono-, di-, and tri-O-acetylated sialic acids on human cells was demonstrated by using radiochromatographic and chemical techniques. Human melanoma cells and fresh colon tissue were biosynthetically labeled with 6- (3H) glucosamine. Radiolabeled sialic acids were hydrolytically removed from cellular glycoconjugates, purified by ion-exchange chromatography, and separated by paper chromatography on the basis of the number of O-substitutions on each sialic molecule. This analytical technique characterized radiolabeled sialic acids that migrated with the same Rf as synthetic mono-, di-, and tri-O-acetylated 14C-labeled sialic acids. The mono-O-acetylated sialic acids were characterized by their sensitivity to sodium periodate oxidation and a crude mouse liver esterase preparation. The di- and tri-O-acetylated sialic acids were characterized by their resistance to sodium periodate oxidation and sensitivity to the action of crude mouse liver esterase. Chromatographically separated di- and tri-O-acetylated sialic acids from normal human colon tissue were characterized by their respective ion molecular weights by using fast-atom bombardment-mass spectrometry. Using these methods, we chemically characterized mono, di-, and tri-O-acetylated sialic acids expressed on human cells. Aberrant expression of O-acetylated sialic acids was associated with adenocarcinoma of the colon, leading to a nearly complete loss of di- and tri-O-acetylated sialic acids.     

High-performance liquid chromatography of long-chain neutral glycosphingolipids and gangliosides

We describe a method for analyzing the perbenzoyl derivatives of both neutral glycosphingolipids and gangliosides with a single high-performance liquid chromatography system. Use of this system, combined with endo- and/or exoglycosidase treatment of glycosphingolipids, provides a sensitive method for obtaining structural information on these compounds. This system has two advantages over previously published chromatography procedures: (i) it uses a commercially available column, and (ii) this single column can be used to analyze gangliosides and their neutral glycosphingolipid products generated by neuraminidase treatment. With this method, we have studied 24 different glycosphingolipids, containing one to ten sugars and one or two sialic acid residues, and have demonstrated its usefulness in evaluating the gangliosides present in human leukocytes.     

Quantitation of picomole levels of N-acetyl- and N-glycolylneuraminic acids by a HPLC-adaptation of the thiobarbituric acid assay

A simple HPLC adaptation of the periodate-TBA assay for free N-acetyl- and N-glycolylneuraminic acids greatly extends the sensitivity and increases the specificity of this standard colorimetric assay. The method, employing a C18 reverse-phase column eluted isocratically with a phosphoric acid-MeOH buffer, is linear over a range of 2 pmol to 20 nmol. Analyses can be performed directly on cell lysates and digests without prior purification of released sialic acids from contaminating salts and biological materials. Interference from 2-deoxysugars is completely eliminated as the chromophore from these compounds is completely resolved from that derived from sialic acids. The application of the technique to quantify cell-surface and total cellular TBA-reactive sialic acids on the surfaces of a variety of tumor cells is described. Additionally, the extent of desialylation of erythrocytes necessary to expose the T antigen is determined.     

环境介质中铂族金属形态分析方法的研究进展

环境中铂族金属(PGMs)的赋存形态多样,形态分析对识别其生态风险具有十分重要的意义。本文综述了环境中3种主要铂族金属(铂、钯、铑)的形态分析方法,包括化学顺序提取、仪器联用技术及计算机模拟等,概述了这些方法的类型、特点及应用,同时阐述了它们存在的不足,并对未来发展方向进行了展望。化学顺序提取法普遍用于固相样品形态分析,当前研究中提出的提取条件和步骤多样,但不能很好地标准化;仪器联用技术在溶液元素形态分析上具有显著优势,毛细管电泳联用系统能够分离具有相同电泳能力的相似物质,但在分离能力和检出限方面不如液相色谱联用系统;计算机模拟则进一步拓展了形态分析的途径,能够实现复杂的形态计算。建议今后将多个方法进行结合,通过相互补充与完善,不断提高分析技术准确性。     

Cryptic sialic acid binding lectins on human blood leukocytes can be unmasked by sialidase treatment or cellular activation.

We recently reported that the sialic acid-specific binding sites of CD22 molecules on B cells are masked by endogenous ligands, and can be unmasked by sialidase treatment or cellular activation. Here, we show that many other human blood leukocyte types have endogenous sialic acid binding sites that can be unmasked by sialidase treatment. Truncation of sialic acid side chains on the soluble probes used for detection abolishes all binding, indicating the specificity of the interaction for the details of sialic acid structure. There is limited overlap between alpha2-6- and alpha2-3-sialic acid-specific binding sites, which are unmasked on monocytes, natural killer cells, a minority of mature T cells, neutrophils, and some cultured human leukemic cell lines. Activation with phorbol ester and calcium ionophore causes spontaneous exposure of some of the binding sites, occurring over a period of minutes on neutrophils and several hours on monocytes and U937 leukemia cells. Activation is accompanied by some evidence for desialylation of cell surface molecules. Thus, many human blood cells have specific binding sites for sialic acids, masked by endogenous sialylated ligands. Cellular activation can unmask these sites, possibly by the action of an endogenous sialidase. The nearly universal masking of such sites in unactivated blood cells could explain why many of these sialic acid-binding lectins have not been previously discovered. Similar considerations may apply to sialic acid binding lectins of other cell types and tissues.