Development of an improved mammalian overexpression method for human CD62L.

We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5-30mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4-6months to produce. To shorten the construction time, we replaced the multi-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ∼5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines.

The adhesive properties of recombinant soluble L-selectin are modulated by its glycosylation.

The leukocyte adhesion molecule L-selectin, which mediates the initial steps of leukocyte attachment to vascular endothelium, is intensely glycosylated. Different glycoforms of L-selectin are expressed on different leukocyte subsets and differences in L-selectin glycosylation appear to be correlated with the leukocyte's ability to attach to different endothelial targets. In the present study we addressed the question whether glycosylation of L-selectin influences L-selectin-ligand interactions. To obtain different glycoforms of L-selectin, recombinant proteins were expressed both in the baby hamster kidney (BHK) cell line and in the human myelogenous cell line K562, resulting in sL-sel[BHK] or sL-sel[K562], respectively. The glycosylation characteristics of the purified proteins were determined. The most striking differences in glycosylation were seen in the terminal sialylation. Each of the two proteins carried sialic acids in the alpha 2-3 position, while alpha 2-6-bound sialic acids were found exclusively on sL-sel[K562]. To investigate their adhesive properties, both recombinant sL-selectins were used in cell adhesion assays and interactions with the ligands present on various hematopoietic cell lines or activated human cardiac microvascular endothelial cells were examined. The binding capacity of sL-sel[K562] was about 1.6 fold higher compared to sL-sel[BHK] under static as well as under flow conditions. These findings indicate that the terminal sialylation pattern of L-selectin modulates its binding characteristics.

Aptamer affinity chromatography: combinatorial chemistry applied to protein purification.

The systematic evolution of ligands by exponential enrichment process is a combinatorial chemistry method that allows the identification of specific oligonucleotide sequences, known as aptamers, that bind to a desired target molecule with high affinity and specificity. Here, a DNA-aptamer specific for human L-selectin was immobilized to a chromatography support to create an affinity column. This column was effectively applied as either the first or second step in the purification of a recombinant human L-selectin-Ig fusion protein from Chinese hamster ovary cell-conditioned medium. The fusion protein was efficiently bound to the column and efficiently eluted by gentle elution schemes. Application of the aptamer column as the initial purification step resulted in a 1500-fold purification with an 83% single step recovery. These results demonstrate that oligonucleotide aptamers can be effective affinity purification reagents.

Identification and characterization of ligands for L-selectin in the kidney. I. Versican, a large chondroitin sulfate proteoglycan, is a ligand for L-selectin.

Ligands for a leukocyte adhesion molecule, L-selectin, are expressed not only in the specific vascular endothelium in lymph nodes and Peyer's patches but also in the extravascular tissues such as the brain white matter, choroid plexus and the kidney distal straight tubuli. However, the biological significance of these extravascular ligands is currently unknown. We now report the purification and characterization of a novel extravascular ligand for L-selectin in the kidney using a tubule-derived cell line, ACHN. Binding of L-selectin-IgG chimera (LEC-IgG) to the isolated ligand was specifically blocked with either (i) anti-L-selectin mAb, (ii) EDTA, (iii) fucoidan, (iv) chondroitin sulfate (CS) B or CS E, or (v) treatment with chondroitinases. Partial amino acid sequencing, Western blotting and immunoprecipitation analyses showed that a major ligand for L-selectin in ACHN cells is versican of 1600 kDa. Histochemical as well as biochemical analyses verified that a versican subspecies in the kidney was indeed reactive with L-selectin. Studies with cell lines including those derived from the kidney indicated that a certain glycoform and/or splice form of versican is reactive with L-selectin. Under pathological conditions such as those induced by unilateral ureteral obstruction, versican was shed from the distal straight tubuli and became localized in the adjacent vascular bundles around which a substantial leukocyte infiltration was concomitantly observed. Possible involvement of versican in leukocyte trafficking into the kidney under diseased conditions is discussed.

Calmodulin regulates L-selectin adhesion molecule expression and function through a protease-dependent mechanism.

Expression of the L-selectin adhesion molecule is rapidly down-regulated upon cell activation through proteolysis at a membrane-proximal site. Here we demonstrate that calmodulin, an intracellular calcium regulatory protein, specifically coprecipitates with L-selectin through a direct association with the cytoplasmic domain of L-selectin. Furthermore, calmodulin inhibitors disrupt L-selectin-dependent adhesion by inducing proteolytic release of L-selectin from the cell surface. The effects of the calmodulin inhibitors on L-selectin expression and function can be prevented by cotreatment with a hydroxamic acid-based metalloprotease inhibitor. Our results suggest a novel role for calmodulin in regulating the expression and function of an integral membrane protein through a protease-dependent mechanism. These findings may have broader implications for other cell surface proteins that also undergo regulated proteolysis.

Affinity and kinetic analysis of L-selectin (CD62L) binding to glycosylation-dependent cell-adhesion molecule-1.

The selectin family of cell adhesion molecules mediates the tethering and rolling of leukocytes on blood vessel endothelium. It has been postulated that the molecular basis of this highly dynamic adhesion is the low affinity and rapid kinetics of selectin interactions. However, affinity and kinetic analyses of monomeric selectins binding their natural ligands have not previously been reported. Leukocyte selectin (L-selectin, CD62L) binds preferentially to O-linked carbohydrates present on a small number of mucin-like glycoproteins, such as glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), expressed in high endothelial venules. GlyCAM-1 is a soluble secreted protein which, following binding to CD62L, stimulates beta2-integrin-mediated adhesion of lymphocytes. Using surface plasmon resonance, we show that a soluble monomeric form of CD62L binds to purified immobilized GlyCAM-1 with a dissociation constant (Kd) of 108 microM. CD62L dissociates from GlyCAM-1 with a very fast dissociation rate constant (>/=10 s-1) which agrees well with the reported dissociation rate constant of CD62L-mediated leukocyte tethers. The calculated association rate constant is >/=10(5) M-1 s-1. At concentrations just above its mean serum level (approximately 1.5 microg/ml or approximately 30 nM), GlyCAM-1 binds multivalently to immobilized CD62L. It follows that soluble GlyCAM-1 may cross-link CD62L when it binds to cells, suggesting a mechanism for signal transduction.

Quantitation of L-selectin distribution on human leukocyte microvilli by immunogold labeling and electron microscopy.

L-Selectin is a leukocyte cell adhesion receptor that contributes to neutrophil (PMN) rolling on activated endothelium at sites of inflammation and mediates lymphocyte attachment to high endothelial venules in peripheral lymph nodes. Localization of this receptor to the tips of PMN and lymphocyte microvilli has been demonstrated. However, its distribution on these cells has not been quantified, and its localization on other leukocytes and the morphometry of microvilli on different leukocyte subpopulations have not been previously examined. In this study, PMN and mononuclear leukocytes were isolated from anticoagulated blood by dextran sedimentation and density centrifugation, fixed in 2% paraformaldehyde and 0.05% glutaraldehyde, immunogold-labeled for L-selectin, and embedded in Epon resin. The distribution of L-selectin was determined by counting gold particles on the plasma membrane of sectioned cells, and the surface microstructure of these cells was surveyed on two-dimensional transmission electron micrographs. On average, 78% of PMN, 72% of monocyte, and 71% of lymphocyte L-selectin was observed on the microvilli, with more variance on lymphocytes than the other cell types. Typical PMN and monocyte sections had 26 microvilli, whereas typical lymphocyte sections had 23. Quantitation of the distribution of L-selectin and leukocyte surface topology offers a foundation from which to study the requirement of microvilli or microvillus-localized L-selectin for leukocyte tethering and rolling in model systems that mimic microvascular environments.

Type IV collagen-binding proteins of neutrophils: possible involvement of L-selectin in the neutrophil binding to type IV collagen.

To isolate type IV collagen-binding proteins, 125I-labeled human-neutrophil extracts were chromatographed on a type IV collagen-Sepharose column. The affinity chromatography-separated fraction contained the four radioactive proteins with apparent molecular masses of 28, 49, 67, and 95 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis indicated that the 95-kD proteins contained both L-selectin and nonspecific cross-reacting antigen 90 (NCA90), and that the 67-kD protein was the 67-kD elastin/laminin-binding protein (67BP). The data obtained with the type IV collagen-affinity chromatography and the immunoaffinity chromatographies using anti-L-selectin and anti-NCA90 monoclonal antibodies (MoAbs) have shown that L-selectin is closely associated with 67BP and the 49-kD protein, and that NCA90 is associated with 67BP, the 28-kD and 49-kD proteins. Among these binding proteins, sialic acid residues were contained in 67BP, L-selectin, and NCA90, but not in the 28-kD and 49-kD proteins. Sialidase treatment completely abolished both the binding affinity of the type IV collagen-binding proteins to type IV collagen and the neutrophil adherence to type IV collagen-coated plastic. Thus, the sialic acid residues of 67BP, L-selectin, and NCA90 seem to be important for the binding of neutrophils to type IV collagen. Furthermore, L-selectin IgG chimeric protein directly bound to type IV collagen-Sepharose column, and anti-L-selectin MoAb DREG56 inhibited the neutrophil adherence to type IV collagen-coated plastic by 51%. These observations suggest that L-selectin likely plays a role in the neutrophil binding to type IV collagen, although neutrophils have several kinds of adhesion molecules for type IV collagen such as L-selectin, NCA90, 67BP, and the 28-kD and 49-kD proteins.

[Identification of spermatozoa L-selectin and two potential pellucida ligands]. / Identification d'une L-sélectine spermatique et de 2 ligands pellucidaires potentiels.

At the molecular level, gamete interactions partly depend on zona pellucida-glycoproteins fucosylation. We show, by immunocytochemistry, the sialyl-lewisx and sialyl-lewisa oligosaccharides on human zonae pellucidae. These epitopes are potential ligands of cell adhesion molecules named selectins which are known to play a role in endothelium-leukocyte interactions. By immunofluorescence, we find the leukocyte selectin (L-selectin) on the spermatozoa head. Preincubation of spermatozoa with an anti-L-selectin monoclonal antibody produces a significant inhibition of zona pellucida tight binding, under hemizona assay conditions. In contrast, preincubation of the zonae pellucidae with anti-sialyl-lewisx or anti-sialyl-lewisa antibodies does not produce a significant inhibition of spermatozoa binding. Western blot analysis of spermatozoa-detergent extracts revealed a band at approximatively 90 kDa with the anti-L-selectin monoclonal antibody. This spermatozoa selectin could play a role in human spermatozoa-zona pellucida binding. Zona-ligands have yet to be precisely defined.