Role of carbohydrate structures in CEA-mediated intercellular adhesion

Human carcinoembryonic antigen (CEA) is a member of a family of cell surface glycoproteins representing a subset of the immunoglobulin superfamily and is a major tumor marker. CEA has been demonstrated to function in vitro, at least, as a homotypic intercellular adhesion molecule. CEA can also inhibit the differentiation of several different cell types and contribute to tumorigenesis, an activity that requires CEA-CEA interactions. Post-translational modifications that could modulate CEA-CEA binding are therefore of interest. CEA is heavily glycosylated with 28 consensus sites for the addition of asparagine-linked carbohydrate structures, leading to a molecule with a bottle brush-like structure. In order to modulate the glycosylation of CEA, we transfected the functional cDNA of CEA into Chinese hamster ovary (CHO) mutant cells, Lec1, Lec2, and Lec8, which are deficient in enzymes responsible for various steps in the glycosylation processing pathway. Aggregation assays of cells in suspension were performed with stable CEA transfectants of these cell lines and showed that all of the aberrant CEA glycoforms could still mediate adhesion. In addition, the specificity of adhesion of these glycoforms was unchanged, as shown by homotypic and heterotypic adhesion assays between the transfectants. Lec1 and Lec2 transfectants did, however, show an increased speed and final extent of aggregation, which is consistent with models in which sugar structures interfere with binding through protein domains. Lec8 transfectants, on the other hand, with more truncated sugar structures than Lec2, showed less aggregation than wild type (WT) transfectants. We therefore conclude that carbohydrates do not determine the adhesion property of CEA or its specificity, in spite of the unusually high degree of glycosylation; they do, however, modulate the strength of adhesion.     

Soluble intercellular adhesion molecules, soluble vascular cell adhesion molecules, and risk of coronary heart disease

Objective: We examined the association of circulating levels of soluble intercellular adhesion molecules (sICAM‐1) and soluble vascular cell adhesion molecules (sVCAM‐1) with coronary heart disease (CHD) risk factors and whether the adhesion molecules alone, and in combination, can serve as predictors of coronary CHD. Research Methods and Procedures: Among 18,225 men from the Health Professional Follow‐up Study who provided blood in 1994, we documented 266 incidents of non‐fatal myocardial infarction or fatal CHD during 6 years of follow‐up. The cases were matched 1:2 with non‐cases on age, smoking, and month of blood draw. We found both adhesion molecules directly associated with BMI, inflammatory biomarkers, and triglycerides and inversely associated with high‐density lipoprotein and alcohol intake (p < 0.05). After adjustment for C‐reactive protein, cholesterol‐to‐high‐density lipoprotein ratio, age, smoking, BMI, physical activity, alcohol intake, history of diabetes, parental history of CHD, aspirin use, antihypertensive drug use, and fasting status, the relative risk of CHD was 1.69 [95% confidence interval (CI), 1.14 to 2.51] for sICAM‐1 and 1.34 (95% CI, 0.91 to 1.96) for sVCAM‐1, when comparing the top quintile with the lower four quintiles. Control for other inflammatory or lipid biomarkers did not appreciably attenuate the associations. When we cross‐classified participants based on their sICAM‐1 and sVCAM‐1 levels, only the men in the top quintile of both biomarkers [relative risk = 2.39 (95% CI, 1.45 to 3.91)] had a significantly elevated risk of CHD (P interaction = 0.01, multivariate model). Discussion: sICAM‐1 and sVCAM‐1 are directly associated with obesity and other CHD risk factors. The combination of high levels of both adhesion molecules might be associated with the development of CHD, independent of other CHD risk factors.     

A direct interaction of axonin-1 with NgCAM-related cell adhesion molecule (NrCAM) results in guidance, but not growth of commissural axons

An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) was shown to play a crucial role in commissural axon guidance across the midline of the spinal cord. We now provide evidence that axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grew preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference was abolished in the presence of anti-axonin-1 antibodies without a decrease in neurite length. Consistent with these findings, commissural axons in vivo only fail to extend along the longitudinal axis when both NrCAM and NgCAM interactions, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, we conclude that axonin-1 is involved in guidance of commissural axons without promoting their growth.     

Fluorophore-labeled carbohydrate analysis of immunoglobulin fusion proteins: Correlation of oligosaccharide content with in vivo clearance profile

CTLA4 is a membrane receptor on cytotoxic T cells whose interaction with the B7 counterreceptor on B cells is important in alloantigen responses. Soluble recombinant human and murine CTLA4 were produced using either Chinese hamster ovary or NS-0 cell lines. Expression vectors were constructed containing the gene coding for the extracellular domain of CTLA4 fused to either human lgG1 hinge, CH2, and CH3 domains or murine lgG2a hinge, CH2, and CH3 domain genes. These glycoproteins were produced in hollow-fiber or packed-bed-type bioreactors and purified from conditioned media by protein A affinity chromatography. Batches of purified CTLA4lg were analyzed for size, composition, and isoelectric point (pl) patterns by standard protein methods; oligosaccharide and monosaccharide profiles using several carbohydrate specific techniques; and in vivo clearance profiles using a murine model. Significant differences were observed between lots in their pl, clearance, and crbohydrate profiles. Higher overall pl values correlated with accelerated alpha-phase clearance and changes in oligosaccharide composition as determined by lectin binding analysis and electrophoresis of fluorophore-conjugated carbohydrates. Preparations exhibiting slower clearance profiles had oligosaccharides with higher quantities of N-acetylneuraminic acid and were predominantly of an N-linked biantennary complex-type. Conversely, batches with accelerated clearance profiles had less detectable N-acetylneuraminic acid. Oligosaccharides from murine CTLA4lg produced in NS-0 cells had terminal N-glycolylneuraminic acid but no detectable N-acetylneuraminic acid and had concomitant accelerated clearance. These data suggest that the presence and quantity of N-acetylneuraminic acid is an important component in predicting CTLA4lg plasma clearance rates and that production lots can be analyzed for oligosaccharide heterogeneity and sialic acid content by electrophoresis of fluorophore-conjugated carbohydrates. (c) 1995 John Wiley & Sons, Inc.     

脂多糖诱导小鼠脏器中胞间粘附分子-1的表达

为研究脂多糖（lipopolysaccharide,LPS)诱导的内毒素休克小鼠多种脏器中胞间粘附分子-1（intercellu-lar adhesion molecule-1,ICAM-1)表达的差异。用5mg/kgLPS腹腔注射小鼠后,分别采用Western blotting和RT-PCR法检测组织中ICAM-1蛋白和mRNA的表达情况,结果显示,在正常小鼠,ICAM-1蛋白和mRNA的表达在肺中最多,其次是脾脏,在肾脏和肠有少量表达,在肝脏和心脏中未能检出,LPS腹腔注射后6h可诱导小鼠发生内毒素休克,此时,ICAM-1蛋白表达仍以在肺中最多,在肝、脾、心、肾和肠依次减少;其中在肺,肾和脾分别比正常时增加4.5、3.0和1.5倍,而且在正常时不能检出的肝和心中呈现阳性,但在肠中则变化不大,脏器中ICAM-1mRNA亦相应显著增加,上述结果表明,在LPS诱导的内毒素休克小鼠的多种脏器中ICAM-1蛋白和mRNA表达显著增加,脏器间ICAM-1表达上调的差异可能带来内毒素休克时脏器的不同易伤性,抑制ICAM-1的表达可能对内毒素休克的防治有重要的意义。     

The effects of Cryptococcus neoformans-secreted antigens on tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 expression on human lung epithelial cells

Since primary infection with Cryptococcus neoformans usually occurs in the lungs, and since pulmonary cryptococcosis involves interactions between yeasts and alveolar epithelial cells, we have begun to study the effects of C. neoformans and its secreted antigens (SA) on epithelial reactions potentially associated with localized inflammation. We report here that SAs from encapsulated and acapsular strains of C. neoformans caused significant reductions in tumor necrosis factor-alpha (TNF-alpha)-induced intercellular adhesion molecule-1 (ICAM-1) expression on A549 lung epithelial cells in culture. We also present evidence that the reduction in ICAM-1 expression was not associated with SA-induced shedding of this adhesion molecule.     

Current Views on Structure and Function of Endothelial Adhesion Molecules

The consecutive events that occur in a living body following injury are commonly referred to as inflammation (inflammare: to set on fire). In the first century A.D. observations of clinical patients had allowed Cornelius Celsus to formulate his famous “cardinal signs” of inflammation: calor, rubor, tumor and dolor. These still holds true today. The four characteristics of inflammation are redness and swelling with heat and pain. From these early studies it was obvious that blood vessels played an important role in the development of an inflammatory process, which coincided with plasma leakage and accumulation of leucocytes in extravascular tissue.     

Current Views on Structure and Function of Endothelial Adhesion Molecules

The consecutive events that occur in a living body following injury are commonly referred to as inflammation (inflammare: to set on fire). In the first century A.D. observations of clinical patients had allowed Cornelius Celsus to formulate his famous “cardinal signs” of inflammation: calor, rubor, tumor and dolor. These still holds true today. The four characteristics of inflammation are redness and swelling with heat and pain. From these early studies it was obvious that blood vessels played an important role in the development of an inflammatory process, which coincided with plasma leakage and accumulation of leucocytes in extravascular tissue.     

Effect of irradiation-induced intercellular adhesion molecule-1 expression on natural killer cell-mediated cytotoxicity toward human cancer cells

Background aims

Irradiation enhances the adhesion between natural killer (NK) cells and target cells by up-regulating intercellular adhesion molecule-1 (ICAM-1) on target cells. Therefore, we investigated the effect of irradiation-induced ICAM-1 expression on human cancer cells on NK cell–mediated cytotoxicity.

The clinical value of assaying maternal serum and amniotic fluid intercellular adhesion molecule 1 (ICAM-1) in cases of premature rupture of membranes

Intercellular adhesion molecule 1 (ICAM-1) expression and upregulation induced by pro-inflammatory cytokines may be of interest in defining human response to inflammation and infection. This study was initiated to determine the levels of ICAM-1 in sera and amniotic fluid of cases of premature rupture of membranes (PROM). Serum and amniotic fluid ICAM-1 levels were determined by ELISA in 33 cases of PROM and 10 cases of normal pregnancies of matched gestational age (controls). Both serum and amniotic fluid ICAM levels were significantly elevated in 76% and 85% of cases of PROM with mean fold increments of 3.13 and 3.95, respectively. This elevation was associated with intra-amniotic infection which was detected by microbiological culture and histopathological evidence of chorioamnionitis. Increased ICAM-1 in cases of PROM may be attributed to neutrophil activation and ICAM-1 expression on fetal membranes and mononuclear cells of amniotic fluid. These results demonstrate that determination of ICAM-1 may be a valuable biomarker for early detection of acute chorioamnionitis and the possibility of PROM.     

N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.     

Low glucose availability alters the expression of genes involved in initial adhesion of human glioblastoma cancer cell line SF767

Studying the metabolic pathways of cancer cells is considered as a key to control cancer malignancies and open windows for effective drug discovery against cancer. Of all the properties of a tumor, metastasis potential is a defining characteristic. Metastasis is controlled by a variety of factors that directly control the expression of cell adhesion proteins. In this study we have investigated the expression of cell to cell and cell to matrix adhesion protein genes during the initial phases of attachment of human glioblastoma cancer cell line SF767 (66Y old human female: UCSF Neurosurgery Tissue Bank) to the attachment surface under (Cell culture treated polystyrene plate bottom) glucose-rich and glucose-starved conditions. The aim was to imitate the natural microenvironment of glucose availability to cancer cells inside a tumor that triggers epithelial to mesenchymal transition (EMT). In this study, we have observed the gene expression of epithelial and mesenchymal isoforms of cadherin (E-CAD and N-CAD) and Ig like cell adhesion molecules (E-CAM and N-CAM) along with Integrin family subunits for the initial attachment of cancer cells. We observed that high glucose environments promoted cell survival and cell adhesion, whereas low glucose accelerated EMT by downregulating the expression level of integrin, E-CAD, and N-CAD, and upregulation of N-CAM during early period of cell adhesion. Low glucose availability also downregulated variety of structural and regulatory genes, such as zinc finger E-box binding home box 1A), cytokeratin, Snail, and β catenin, and upregulation of hypoxia-inducible factor 1, matrix metalloprotease 13/Collagenase 3, vimentim, p120, and fructose 1,6 bisphosphatase. Glucose conditions are more efficient for cancer studies in this case glioblastoma cells.     

Endothelial intercellular cell adhesion molecule 1 contributes to cell aggregate formation in CHO cells cultured in serum-free media

Chinese hamster ovary (CHO) cells have been adapted to grow in serum-free media and in suspension culture to facilitate manufacturing needs. Some CHO cell lines, however, tend to form cell aggregates while being cultured in suspension. This can result in reduced viability and capacity for single cell cloning (SCC) via limiting dilution, and process steps to mitigate cell aggregate formation, for example, addition of anti-cell-aggregation agents. In this study, we have identified endothelial intercellular cell adhesion molecule 1 (ICAM-1) as a key protein promoting cell aggregate formation in a production competent CHO cell line, which is prone to cell aggregate formation. Knocking out (KO) the ICAM-1 gene significantly decreased cell aggregate formation in the culture media without anti-cell-aggregation reagent. This trait can simplify the process of transfection, selection, automated clone isolation, and so on. Evaluation in standard cell line development of ICAM-1 KO and wild-type CHO hosts did not reveal any noticeable impacts on titer or product quality. Furthermore, analysis of a derived nonaggregating cell line showed significant reductions in expression of cell adhesion proteins. Overall, our data suggest that deletion of ICAM-1 and perhaps other cell adhesion proteins can reduce cell aggregate formation and improve clonality assurance during SCC.     

The cloning and preliminarily functional analysis of the human neurotrimin gene

~~The cloning and preliminarily functional analysis of the human neurotrimin gene~~     

Cytoplasmic domain is not essential for the cell adhesion activities of gicerin, an Ig-superfamily molecule

Gicerin is a cell adhesion molecule in the immunoglobulin (Ig) superfamily and is expressed abundantly during development in the nervous system. It has homophilic cell adhesion activity and also has heterophilic binding activity with NOF (neurite outgrowth factor) and mediates neurite extension. There are two isoforms of gicerin, one with a short (s-gicerin) and the other with a longer cytoplasmic domain (l-gicerin). We have reported that s-gicerin possesses stronger activities than l-gicerin during cell aggregation, in NOF-binding, and in neurite extension. In this study, we established cell lines which expressed a mutant-gicerin whose cytoplasmic domain was deleted and we compared the above three biological activities of the mutant-gicerin with those of s- and l-gicerin. We found that the mutant-gicerin retained all these activities, but the activities were weaker than those of s-gicerin and almost the same as those of l-gicerin. We concluded that the cytoplasmic domain of gicerin is not essential for optimal adhesive activities of gicerin, but might be involved in the regulation of its activities.     

Expression and involvement of gicerin, a cell adhesion molecule, in the development of chick optic tectum

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It has both a homophilic binding activity and a heterophilic binding activity to neurite outgrowth factor (NOF) a molecule belonging to the laminin family. We have reported many studies on the heterophilic activity of gicerin and NOF, but the function of its homophilic binding activity in vivo had been unclear. In the retina, gicerin is expressed in retinal ganglion cells only when they extend neurites to the optic tectum. In this report we have found that gicerin is also transiently expressed in the optic tectum during this time. First, cell aggregation assays were used to show that gicerin expressed in the optic tectum displays homophilic binding activity. Then, explant cultures of embryonic day 6 chick optic tectum on gicerin-Fc chimeric protein-coated dishes and NOF-coated dishes were carried out. It was found that gicerin-gicerin homophilic interactions promoted cell migration, whereas heterophilic interactions with NOF induced neurite formation. Furthermore, when anti-gicerin antibodies were injected in order to examine the effect of gicerin protein in the formation of the tectal layer in ovo, cell migration was strongly inhibited. These data suggest that homophilic interaction of gicerin participates in the migration of neural cells during the layer formation and plays a crucial role in the organization of the optic tectum.     

Critical and optimal Ig domains for promotion of neurite outgrowth by L1/Ng‐CAM

Mammalian L1 and avian Ng‐CAM are homologous neural cell adhesion molecules (CAMs) that promote neurite outgrowth and cell adhesion in most neurons. Previous attempts to map these activities to discrete regions in the CAMs have suggested the involvement of a variety of different domains. However, these studies mainly used bacterially expressed proteins that were much less active on a molar basis than the native molecules. To define regions that are critical for maximal neurite outgrowth, we constructed and tested a panel of eukaryotically expressed proteins containing various extracellular segments of human L1 (hL1) or Ng‐CAM. Our results indicate that Ig domains 1–4 of hL1 are critical for homophilic binding and neurite outgrowth; however this segment is less potent than the entire extracellular region. Optimal neurite outgrowth activity was seen with proteins containing all six Ig domains of hL1 or Ng‐CAM. The adhesive properties of hL1 fragments correlated tightly with their neurite outgrowth activities, suggesting that these two processes are closely linked. These results suggest that Ig domains 1–4 form a structural cassette responsible for hL1 homophilic binding, while Ig domains 1–6 represent a functional region for optimal promotion of neurite outgrowth in vitro and possibly in vivo. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 287–302, 2000     

The UNC-112 gene in Caenorhabditis elegans encodes a novel component of cell-matrix adhesion structures required for integrin localization in the muscle cell membrane

Embryos homozygous for mutations in the unc-52, pat-2, pat-3, and unc-112 genes of C. elegans exhibit a similar Pat phenotype. Myosin and actin are not organized into sarcomeres in the body wall muscle cells of these mutants, and dense body and M-line components fail to assemble. The unc-52 (perlecan), pat-2 (alpha-integrin), and pat-3 (beta-integrin) genes encode ECM or transmembrane proteins found at the cell-matrix adhesion sites of both dense bodies and M-lines. This study describes the identification of the unc-112 gene product, a novel, membrane-associated, intracellular protein that colocalizes with integrin at cell-matrix adhesion complexes. The 720-amino acid UNC-112 protein is homologous to Mig-2, a human protein of unknown function. These two proteins share a region of homology with talin and members of the FERM superfamily of proteins.We have determined that a functional UNC-112::GFP fusion protein colocalizes with PAT-3/beta-integrin in both adult and embryonic body wall muscle. We also have determined that UNC-112 is required to organize PAT-3/beta-integrin after it is integrated into the basal cell membrane, but is not required to organize UNC-52/perlecan in the basement membrane, nor for DEB-1/vinculin to localize with PAT-3/beta-integrin. Furthermore, UNC-112 requires the presence of UNC-52/perlecan and PAT-3/beta-integrin, but not DEB-1/vinculin to become localized to the muscle cell membrane.