A benzoboroxole‐based affinity ligand for glycoprotein purification at physiological pH

Developing ligands capable of carbohydrate recognition has become increasingly important as the essential roles of glycoproteins and glycolipids in a diverse array of cellular signaling, pathophysiology, and immune response mechanisms are elucidated. Effective ligands for the glycan portion of glycoproteins and glycolipids are needed for pre‐enrichment proteomics strategies, as well as for the purification of individual glycoproteins from complex biological milieu encountered both in biochemistry research and bio‐pharmaceutical development. In this work, we developed a carbohydrate specific affinity ligand for glycoprotein purification using a one‐pot, multi‐component synthesis reaction (Ugi synthesis) and an amine‐functionalized benzoboroxole moiety immobilized on agarose beads. Benzoboroxoles are unique boronic acid derivatives that have recently been found to bind specifically to the cis‐diol groups of carbohydrates at physiological pH, with superior affinity to any other Wulff‐type boronic acid. The solid‐phase affinity ligand developed herein specifically binds the carbohydrate moiety of the glycoprotein glucose oxidase, as well as a fluorescein isothiocyanate‐dextran, as shown through deglycosylation binding studies. Additionally, the ligand is able to purify glucose oxidase from crude Escherichia coli lysate, at physiological pH, equitably to commercially available boronic acid‐functionalized agarose beads that required alkaline pH conditions. Thus, this affinity ligand is a marked improvement on current, commercially available boronic acid‐based glycoprotein enrichment matrices and has the potential to exhibit high individual glycoprotein specificity because of the additional functional groups available for variation on the Ugi scaffold. Copyright © 2015 John Wiley & Sons, Ltd.     

Alkaline treatment has contrasting effects on the structure of deglycosylated and glycosylated forms of glucose oxidase

X-ray crystallographic studies on glucose oxidase showed a strong interaction between carbohydrate and protein moieties of the glycoprotein. However, experimental studies under physiological conditions reported no influence of carbohydrate moiety on the structural and functional properties of glucose oxidase. In order to demonstrate the role of carbohydrate moiety on the structure and stability, we carried out a detailed comparative study on the pH-induced structural changes in the native and deglycosylated forms of glucose oxidase. Our studies demonstrate that at physiological pH both forms of enzyme have very similar structural and stability properties. Acid denaturation also showed similar structural changes in both forms of the enzyme. However, on alkaline treatment contrasting effects on the structure and stability of the two forms of enzyme were observed. The glycosylated enzyme undergoes partial unfolding with decreased stability at alkaline pH; however, a compaction of native conformation and enhanced stability of enzyme was observed for the deglycosylated enzyme under similar conditions. This is the first experimental demonstration of the influence of carbohydrate moiety on structure and stability of glucose oxidase. The studies also indicate the importance of pH studies in evaluating the effect of carbohydrate moiety on the structural and stability properties of glycoprotein.     

An artificial receptor for glycoproteins

We describe the rational design, synthesis and development of a sterilizable biomimetic ligand for the affinity purification of glycoproteins. Based on mimicking the principles of natural carbohydrate recognition, a putative library of 196 glycoprotein-binding synthetic ligands was designed and synthesized on a polymeric support. Ligand 11/11, based on a triazine scaffold and immobilized on a hydrophilic support, was identified as the "lead" ligand. The carbohydrate recognizing the potential of the "lead" ligand was revealed by reduced binding of a periodate oxidized model glycoprotein, and by "sharp" elution profiles achieved with borate buffer eluents. Specific elution and competitive binding experiments determined the monosaccharide specificity of 11/11 in the order mannoside > glucoside > galactoside. The diastereo-selective performance of ligand 11/11 was quantified and reaffirmed by analytical affinity chromatography and (1)H-NMR, in the order galactoside < glucoside < mannoside with binding affinities (K(a), M(-1)) in the 63-214 and 20-83 M(-1) range, respectively. Partition coefficient analysis revealed binding constants towards glycoproteins in the 10(4) M(-1) range, that compared favourably with the affinities of carbohydrate binding lectins for glycoproteins, such as concanavalin A. Molecular modelling studies of ligand 11/11 revealed the formation of a pre-organized apolar "tweezer-like" cavity, containing complementary nitrogenous hydrogen bond donor and acceptor groups that formed selective interactions with the equatorial 3- and 4-hydroxyl groups of saccharides.     

O-linked protein glycosylation structure and function

There has been a recent resurgence of interest in the post-translational modification of serine and threonine hydroxyl groups by glycosylation, because the resulting O-linked oligosaccharide chains tend to be clustered over short stretches of peptide and hence they can present multivalent carbohydrate antigenic or functional determinants for antibody recognition, mammalian cell adhesion and microorganism binding. Co-operativity can greatly increase the affinity of interactions with antibodies or carbohydrate binding proteins. Thus, in addition to their known importance in bearing tumour associated antigens in the gastrointestinal and respiratory tracts, glycoproteins with O-linked chains have been implicated as ligands or co-receptors for selectins (mammalian carbohydrate binding proteins). Microorganisms may have adopted similar mechanisms for interactions with mammalian cells in infection, by having relatively low affinity ligands (adhesins) for carbohydrate binding, which may bind with higher affinity due to the multivalency of the host ligand and which are complemented by other virulence factors such as interactions with integrin-type molecules. In addition to specific adhesion signals from O-linked carbohydrate chains, multivalent O-glycosylation is involved in determining protein conformation and forming conjugate oligosaccharide-protein antigenic, and possible functional determinants.     

Synthesis and screening of a rationally designed combinatorial library of affinity ligands mimicking protein L from Peptostreptococcus magnus

Rational design and combinatorial chemistry were utilized to search for lead protein L (PpL) mimetics for application as affinity ligands for the purification of antibodies and small fragments, such as Fab and scFv, and as potential diagnostic or therapeutic agents. Inspection of the key structural features of the complex between PpL and human Fab prompted the de novo design and combinatorial synthesis of a 169-membered solid-phase ligand library, which was assessed for binding to human IgG and subsequent selectivity for the Fab fragment. Eight ligands were selected, chemically characterized and compared with a commercial PpL-adsorbent for binding pure immunoglobulin fractions. The most promising lead, ligand 8/7, when immobilized on an agarose support, behaved in a similar fashion to PpL in isolating Fab fragments from papain digests of human IgG to a final purity of 97%.     

Monospecific and common glycoprotein ligands for E- and P-selectin on myeloid cells

E- and P-selectin are inducible cell adhesion molecules on endothelial cells, which function as Ca(2+)-dependent lectins and mediate the binding of neutrophils and monocytes. We have recently identified a 150- kD glycoprotein ligand for E-selectin on mouse myeloid cells, using a recombinant antibody-like form of mouse E-selectin. Here, we report that this ligand does not bind to an analogous P-selectin fusion protein. Instead, the chimeric P-selectin-IgG protein recognizes a 160- kD glycoprotein on the mouse neutrophil progenitor 32D cl 3, on mature mouse neutrophils and on human HL60 cells. The binding is Ca(2+)- dependent and requires the presence of sialic acid on the ligand. This P-selectin-ligand is not recognized by E-selectin. Removal of N-linked carbohydrate side chains from the 150-kD and the 160-kD monospecific selectin ligands abolishes the binding of both ligands to the respective selectin. Treatment of HL60 cells with Peptide: N- glycosidase F inhibited cell binding to P- and E-selectin. In addition, glycoproteins of 230 and 130 kD were found on mature mouse neutrophils, which bound both to E- and P-selectin in a Ca(2+)-dependent fashion. The signals detected for these ligands were 15-20-fold weaker than those for the monospecific ligands. Both proteins were heavily sialylated and selectin-binding was blocked by removal of sialic acid, but not by removal of N-linked carbohydrates. Our data reveal that E- and P-selectin recognize two categories of glycoprotein ligands: one type requires N-linked carbohydrates for binding and is monospecific for each of the two selectins and the other type binds independent of N- linked carbohydrates and is common for both endothelial selectins.     

Role of a vitelline layer-associated 350 kDa glycoprotein in controlling species-specific gamete interaction in the sea urchin

The process of sperm-egg binding is one of the barriers to cross-fertilization between related sea urchin species. A 350 kDa glycoprotein in the egg vitelline layer of Strongylocentrotus purpuratus has been shown to be a sperm-binding protein (SBP). Sulfated O-linked oligosaccharide chains on the 350 kDa glycoprotein, as well as domains of the polypeptide chain, serve as ligands for this binding process. The hypothesis that species-specific sperm-egg binding is attributed to the interaction between the sperm and the 350 kDa glycoprotein was tested using S. purpuratus and S. franciscanus. It was found that both species had a 350 kDa glycoprotein on the egg surface that cross-reacted immunologically using antibodies prepared against a recombinant form of the SBP. Because earlier studies had implicated the carbohydrate chains of the 350 kDa glycoprotein of S purpuratus in sperm binding, differences in carbohydrate chains on the 350 kDa glycoproteins of these species were examined. It was found that among the lectins tested only wheat germ agglutinin and Sambucus nigra agglutinin showed a significant difference in reactivity to the 350 kDa glycoproteins between species. Finally, using a bead-binding assay, it was shown that the isolated 350 kDa glycoproteins exhibited species-specific sperm-binding activity.     

Identification of neutrophil granule glycoproteins as Lewis(x)-containing ligands cleared by the scavenger receptor C-type lectin

The scavenger receptor C-type lectin (SRCL) is a glycan-binding receptor that has the capacity to mediate endocytosis of glycoproteins carrying terminal Lewis(x) groups (Galβ1-4(Fucα1-3)GlcNAc). A screen for glycoprotein ligands for SRCL using affinity chromatography on immobilized SRCL followed by mass spectrometry-based proteomic analysis revealed that soluble glycoproteins from secondary granules of neutrophils, including lactoferrin and matrix metalloproteinases 8 and 9, are major ligands. Binding competition and surface plasmon resonance analysis showed affinities in the low micromolar range. Comparison of SRCL binding to neutrophil and milk lactoferrin indicates that the binding is dependent on cell-specific glycosylation in the neutrophils, as the milk form of the glycoprotein is a much poorer ligand. Binding to neutrophil glycoproteins is fucose-dependent, and mass spectrometry-based glycomic analysis of neutrophil and milk lactoferrin was used to establish a correlation between high affinity binding to SRCL and the presence of multiple clustered terminal Lewis(x) groups on a heterogeneous mixture of branched glycans, some with poly N-acetyllactosamine extensions. The ability of SRCL to mediate uptake of neutrophil lactoferrin was confirmed using fibroblasts transfected with SRCL. The common presence of Lewis(x) groups in granule protein glycans can thus target granule proteins for clearance by SRCL. PCR and immunohistochemical analysis confirm that SRCL is widely expressed on endothelial cells and thus represents a distributed system that could scavenge released neutrophil glycoproteins both locally at sites of inflammation or systemically when they are released in the circulation.     

Terminal alpha-linked galactose rather than N-acetyl lactosamine is ligand for bovine heart galectin-1 in N-linked oligosaccharides of glycoproteins

Preference for the beta-anomer of galactose attributed to the bovine heart 14 kDa galectin-1 (BHL-14) was re-examined using natural glycoproteins and artificially glycosylated proteins as ligands. Endogenous glycoproteins co-purified with BHL-14 during its affinity chromatographic isolation contained oligosaccharides bearing terminal alpha-linked galactose (TAG) moieties and were superior even to laminin as ligands for homogeneous BHL-14 obtained by high pressure liquid chromatography. Artificially glycosylated proteins prepared by covalent attachment of melibiose to proteins and containing TAG moieties were ligands for BHL-14, unlike their lactose counterparts which contained beta-linked galactose. Enzymatic removal of TAG moieties from the following glycoproteins abolished their recognition by BHL-14: (i) endogenous glycoproteins co-purified with BHL-14; (ii) mouse laminin; and (iii) bovine heart glycoproteins recognized by peanut agglutinin. Modification of TAG in laminin using galactose oxidase also rendered the glycoprotein inert towards BHL-14. Desialylation of human IgG, bovine thyroglobulin or laminin failed to increase the affinity of BHL-14 for these glycoproteins. Since removal of TAG or of sialic acid moiety exposed LacNAc (Gal beta1-->4 GlcNAc) in these glycoproteins, these results indicated that TAG, rather than LacNAc, is a ligand for BHL-14 on N-linked oligosaccharide chains of glycoproteins. Ready recognition of human IgA and jacalin-binding human plasma glycoproteins and non-recognition of human IgG suggested that T antigen (Galbeta1-->3 GalNAc) may also be ligand for galectin-1.     

Combinatorial chemistry defines general properties of linkers for the optimal display of peptide ligands for binding soluble protein targets to TentaGel microscopic beads

Affinity chromatography and the binding of soluble target proteins to novel or known ligands attached to solid supports are important phenomena to basic and applied research. Satisfactory display of a ligand for the acceptor protein is critical for successful binding to occur. Here we describe the application of combinatorial chemistry to systematically explore the properties of linkers used to present peptide ligands to various protein targets. Our main interest is in drug discovery, and our results probably explain, in large part, the disappointing efficiency of an early drug discovery method known as the "Selectide Process" (Lam, K. S., et al. (1991) Nature 358, 82-84). Interestingly, for all seven protein targets studied, a cationic feature was found to be a common theme for optimal linkers displaying peptide ligands on TentaGel beads, and this is not likely to be caused by ionic exchange mechanisms.     

An application of CIFAP for predicting the binding affinity of Chk1 inhibitors derived from 2‐aminothiazole‐4‐carboxamide

Investigation of protein‐ligand interactions obtained from experiments has a crucial part in the design of newly discovered and effective drugs. Analyzing the data extracted from known interactions could help scientists to predict the binding affinities of promising ligands before conducting experiments. The objective of this study is to advance the CIFAP (compressed images for affinity prediction) method, which is relevant to a protein‐ligand model, identifying 2D electrostatic potential images by separating the binding site of protein‐ligand complexes and using the images for predicting the computational affinity information represented by pIC₅₀ values. The CIFAP method has 2 phases, namely, data modeling and prediction. In data modeling phase, the separated 3D structure of the binding pocket with the ligand inside is fitted into an electrostatic potential grid box, which is then compressed through 3 orthogonal directions into three 2D images for each protein‐ligand complex. Sequential floating forward selection technique is performed for acquiring prediction patterns from the images. In the prediction phase, support vector regression (SVR) and partial least squares regression are used for testing the quality of the CIFAP method for predicting the binding affinity of 45 CHK1 inhibitors derived from 2‐aminothiazole‐4‐carboxamide. The results show that the CIFAP method using both support vector regression and partial least squares regression is very effective for predicting the binding affinities of CHK1‐ligand complexes with low‐error values and high correlation. As a future work, the results could be improved by working on the pose of the ligands inside the grid.     

Protein stabilization with a dipeptide‐mimic triazine‐scaffolded synthetic affinity ligand

Protein stabilization was achieved by a novel approach based on the adsorption and establishment of affinity‐like interactions with a biomimetic triazine‐scaffolded ligand. A synthetic lead compound (ligand 3′/11, K_a ≈ 10⁴ M^?1) was selected from a previously screened solid‐phase library of affinity ligands for studies of adsorption and stabilization of cutinase from Fusarium solani pisi used as a model system. This ligand, directly synthesized in agarose by a well‐established solid‐phase synthesis method, was able to strongly bind cutinase and led to impressive half‐lives of more than 8 h at 70 °C, and of approximately 34 h at 60 °C for bound protein (a 25‐ and 57‐fold increase as compared with the free enzyme, respectively). The ligand density in the solid matrix was found to be a determinant parameter for cutinase stabilization. It is conceivable that the highly stabilizing effect observed results from the binding of more than one ligand residue to the enzyme, creating specific macromolecular configurations that lock structural mobility thus improving molecular stability. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of multivalent lactose quantum dots and its application in carbohydrate–protein interactions study and cell imaging

We have previously reported a facile and convenient method for the preparation of a new type of lactose-CdSeS/ZnS quantum dots conjugates (Lac-QDs) that exhibit biocompatibility, noncytotoxicity and specificity to leukocytes. In order to further study the carbohydrate–protein interactions, a series of Lac-QDs with different lactose densities and a PEGylated (n = 3) lactose-QDs conjugate (LacPEG-QDs) with more flexible sugar ligands were prepared. The amount of the sugar molecules on QDs can be determined by NMR, which was in agreement with the results from TGA determination. The formula of the conjugates was determined with ICP-OES. The interactions between the conjugated QDs and the PNA protein were measured using SPR, which revealed that higher lactose density favored binding affinity under the same concentration, and Lac-QDs exhibit higher affinity than LacPEG-QDs. We further used a solid phase assay to assess the anti-adhesion activity of Lac-QDs and LacPEG-QDs on the cell level. The results showed that Lac-QDs had stronger activity in preventing THP1 from adhering to HUVEC than LacPEG-QDs, which was consistent with the SPR results. We reasoned that decrease in the conformational entropy induced by appropriate restriction of sugar flexibility could enhance the binding affinity of glyco-QDs, which implies that entropy change may be the main contributor to the interaction between high valent glyco-QDs and protein. The fabrication of lactose on QDs provides a fluorescent multivalent carbohydrate probe that can be used as mimics of glycoprotein for the study of carbohydrate–protein interactions and cell imaging.     

Terminal sialic acids on CD44 N‐glycans can block hyaluronan binding by forming competing intramolecular contacts with arginine sidechains

Specific sugar residues and their linkages form the basis of molecular recognition for interactions of glycoproteins with other biomolecules. Seemingly small changes, like the addition of a single monosaccharide in the covalently attached glycan component of glycoproteins, can greatly affect these interactions. For instance, the sialic acid capping of glycans affects protein‐ligand binding involved in cell–cell and cell–matrix interactions. CD44 is a single‐pass transmembrane glycoprotein whose binding with its carbohydrate ligand hyaluronan (HA), an extracellular matrix component, mediates processes such as leukocyte homing, cell adhesion, and tumor metastasis. This binding is highly regulated by glycosylation of the N‐terminal extracellular hyaluronan‐binding domain (HABD); specifically, sialic acid capped N‐glycans of HABD inhibit ligand binding. However, the molecular mechanism behind this sialic acid mediated regulation has remained unknown. Two of the five N‐glycosyation sites of HABD have been previously identified as having the greatest inhibitory effect on HA binding, but only if the glycans contain terminal sialic acid residues. These two sites, Asn25 and Asn120, were chosen for in silico glycosylation in this study. Here, from extensive standard molecular dynamics simulations and biased simulations, we propose a molecular mechanism for this behavior based on spontaneously‐formed charge‐paired hydrogen bonding interactions between the negatively‐charged sialic acid residues and positively‐charged Arg sidechains known to be critically important for binding to HA, which itself is negatively charged. Such intramolecular hydrogen bonds would preclude associations critical to hyaluronan binding. This observation suggests how CD44 and related glycoprotein binding is regulated by sialylation as cellular environments fluctuate. Proteins 2014; 82:3079–3089. © 2014 Wiley Periodicals, Inc.     

Assembly of cell-wall glycoproteins of Chlamydomonas reinhardii: Oligosaccharides are added in medial and trans Golgi compartments

A series of monoclonal antibodies and a polyclonal antiserum have been used to investigate the localisation and pathway of biosynthesis of the cell-wall hydroxyproline-rich glycoprotein 2BII in the alga Chlamydomonas reinhardii. Glyco-protein precursors were detected within the endoplasmic reticulum using a polyclonal antiserum raised to the deglycosylated 2BII. Monoclonal antibodies which are known to recognise different carbohydrate epitopes of 2BII were found to label two distinct regions of the Golgi stack. The immunolabelling results demonstrate that there is compartmentation of protein synthesis and glycosylation steps for these O-glycosidically linked glycoproteins. Newly synthesised glycoproteins are transported from the Golgi apparatus to the cell surface via two distinct routes. They then undergo assembly into a cell wall, the inner wall layer being formed first and probably functionaing as a template within which the outer crystalline wall layers are assembled.Abbreviations DGP deglycosylated glycoprotein - ER endoplasmic reticulum - MAC monoclonal antibody centre - M _r relative molecular mass     

Tumor biomarker glycoproteins in the seminal plasma of healthy human males are endogenous ligands for DC-SIGN

DC-SIGN is an immune C-type lectin that is expressed on both immature and mature dendritic cells associated with peripheral and lymphoid tissues in humans. It is a pattern recognition receptor that binds to several pathogens including HIV-1, Ebola virus, Mycobacterium tuberculosis, Candida albicans, Helicobacter pylori, and Schistosoma mansoni. Evidence is now mounting that DC-SIGN also recognizes endogenous glycoproteins, and that such interactions play a major role in maintaining immune homeostasis in humans and mice. Autoantigens (neoantigens) are produced for the first time in the human testes and other organs of the male urogenital tract under androgenic stimulus during puberty. Such antigens trigger autoimmune orchitis if the immune response is not tightly regulated within this system. Endogenous ligands for DC-SIGN could play a role in modulating such responses. Human seminal plasma glycoproteins express a high level of terminal Lewis(x) and Lewis(y) carbohydrate antigens. These epitopes react specifically with the lectin domains of DC-SIGN. However, because the expression of these sequences is necessary but not sufficient for interaction with DC-SIGN, this study was undertaken to determine if any seminal plasma glycoproteins are also endogenous ligands for DC-SIGN. Glycoproteins bearing terminal Lewis(x) and Lewis(y) sequences were initially isolated by lectin affinity chromatography. Protein sequencing established that three tumor biomarker glycoproteins (clusterin, galectin-3 binding glycoprotein, prostatic acid phosphatase) and protein C inhibitor were purified by using this affinity method. The binding of DC-SIGN to these seminal plasma glycoproteins was demonstrated in both Western blot and immunoprecipitation studies. These findings have confirmed that human seminal plasma contains endogenous glycoprotein ligands for DC-SIGN that could play a role in maintaining immune homeostasis both in the male urogenital tract and the vagina after coitus.     

Identification of Novel Contributions to High-affinity Glycoprotein-Receptor Interactions using Engineered Ligands

Engineered receptor fragments and glycoprotein ligands employed in different assay formats have been used to dissect the basis for the dramatic enhancement of binding of two model membrane receptors, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the macrophage galactose lectin, to glycoprotein ligands compared to simple sugars. These approaches make it possible to quantify the importance of two major factors that combine to enhance the affinity of single carbohydrate-recognition domains (CRDs) for glycoprotein ligands by 100-to 300-fold. First, the presence of extended binding sites within a single CRD can enhance interaction with branched glycans, resulting in increases of fivefold to 20-fold in affinity. Second, presentation of glycans on a glycoprotein surface increases affinity by 15-to 20-fold, possibly due to low-specificity interactions with the surface of the protein or restriction in the conformation of the glycans. In contrast, when solution-phase networking is avoided, enhancement due to binding of multiple branches of a glycan to multiple CRDs in the oligomeric forms of these receptors is minimal and binding of a receptor oligomer to multiple glycans on a single glycoprotein makes only a twofold contribution to overall affinity. Thus, in these cases, multivalent interactions of individual glycoproteins with individual receptor oligomers have a limited role in achieving high affinity. These findings, combined with considerations of membrane receptor geometry, are consistent with the idea that further enhancement of the binding to multivalent glycoprotein ligands requires interaction of multiple receptor oligomers with the ligands.     

A galactose-rich, cell-wall glycoprotein from styles of Nicotiana alata

A basic, galactose-rich style glycoprotein (GaRSGP) encoded by a previously characterized style-specific cDNA (NaPRP4) has been isolated from the styles of Nicotiana alata and structurally characterized. The glycoprotein is associated with cell walls in the transmitting tract and is composed of approximately 25% (w/w) protein and 75% (w/w) carbohydrate. The purified glycoprotein appears as a smear of between 45–120 kDa on SDS—PAGE; the deglycosylated protein backbone has an apparent molecular weight of approximately 30 kDa. The glycoprotein is rich in the amino acids lysine, proline, and hydroxyproline and in the monosaccharides galactose and arabinose. It is one of only a few proline/hydroxyproline-rich glycoproteins (P/HRGPs) to be characterized both as a cDNA-clone and protein. Glycans are attached to the protein backbone through both O - and N -glycosidic linkages with the majority of the carbohydrate being O -linked and consisting of short, highly branched chains terminating primarily in galactose residues. A carbohydrate epitope(s) is found on both GaRSGP and another style-specific glycoprotein but not on glycoproteins from other tissues. This finding provides further evidence for the existence of a style-specific carbohydrate epitope(s) which may play a role in style function.     

Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence-activated cell sorting

Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N‐linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence‐activated cell sorting (FACS) and site‐specific gene excision to establish high‐yield glycoprotein expression for structural studies with stable clones derived from the well‐established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single‐chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome‐associated membrane protein 3 (LAMP3d). In both cases, stable GFP‐expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.     

L-Selectin from Human, but Not from Mouse Neutrophils Binds Directly to E-Selectin

L-Selectin on neutrophils as well as inducible E- and P-selectin on endothelium are involved in the recruitment of neutrophils into inflamed tissue. Based on cell attachment assays, L-selectin was suggested to function as a carbohydrate presenting ligand for E- and P-selectin. However, previous affinity isolation experiments with an E-selectin–Ig fusion protein had failed to detect L-selectin among the isolated E-selectin ligands from mouse neutrophils. We show here that L-selectin from human neutrophils, in contrast to mouse neutrophils, can be affinity-isolated as a major ligand from total cell extracts using E-selectin–Ig as affinity probe. Binding of human L-selectin to E-selectin was direct, since purified L-selectin could be reprecipitated with E-selectin–Ig. Recognition of L-selectin was abolished by sialidase-treatment, required Ca²⁺, and was resistant to treatment with endoglycosidase F. Binding of L-selectin to a P-selectin–Ig fusion protein was not observed. In agreement with the biochemical data, the anti–Lselectin mAb DREG56 inhibited rolling of human neutrophils on immobilized E-selectin–Ig but not on P-selectin–Ig. No such inhibitory effect was seen with the anti–mouse L-selectin mAb MEL14 on mouse neutrophils. Rolling of E-selectin transfectants on purified and immobilized human L-selectin was inhibited by mAb DREG56. We conclude that L-selectin on human neutrophils is a major glycoprotein ligand among very few glycoproteins that can be isolated by an E-selectin affinity matrix. The clear difference between human and mouse L-selectin suggests that E-selectin–binding carbohydrate moieties are attached to different protein scaffolds in different species.