Differential recognition by conglutinin and mannan-binding protein of N-glycans presented on neoglycolipids and glycoproteins with special reference to complement glycoprotein C3 and ribonuclease B.

Conglutinin and mannan-binding protein are serum proteins that have similar carbohydrate binding specificities toward high mannose-type oligosaccharides, and yet only conglutinin binds the complement glycoprotein iC3b, which contains oligosaccharides of this type. In the present study, the interactions of conglutinin and mannan-binding protein were evaluated with the complement glycoprotein C3, including various physiologically derived fragments of this glycoprotein, and neoglycolipids prepared from oligosaccharides released from C3 and its isolated alpha and beta chains. Several conclusions can be drawn.

Sulfated blood group Lewis(a). A superior oligosaccharide ligand for human E-selectin.

In earlier studies of oligosaccharide probes (neoglycolipids) generated from an ovarian cystadenoma glycoprotein, one of the components that strongly supported binding of the endothelial adhesion molecule, E-selectin, was identified as an equimolar mixture of tetrasaccharides of blood group Le(a) and Le(x) type sulfated at position 3 of the outer galactose (C.-T. Yuen, A.

Differences in the binding specificities of Pseudomonas aeruginosa M35 and Escherichia coli C600 for lipid-linked oligosaccharides with lactose-related core regions.

Membrane glycolipids contain the lactose sequence (galactose linked to glucose), and the oligosaccharide is variously extended such that there is a cell-type-specific repertoire. In this study, binding of Pseudomonas aeruginosa M35 to lipid-linked lactose (Gal beta 1-4Glc [structure 1]), lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc [structure 2]), lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc [structure 3]), and asialo GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc [structure 4]) was evaluated and compared with binding of Escherichia coli C600 to these compounds. Oligosaccharides were linked to the lipid phosphatidylethanolamine dipalmitoate, and the resulting neoglycolipids were resolved on thin-layer chromatograms or coated onto plastic microtiter wells.

High affinity binding of the leucocyte adhesion molecule L-selectin to 3'-sulphated-Le(a) and -Le(x) oligosaccharides and the predominance of sulphate in this interaction demonstrated by binding studies with a series of lipid-linked oligosaccharides.

The binding of the leucocyte adhesion molecule L-selectin has been investigated toward several structurally defined lipid-linked oligosaccharides immobilized on silica gel chromatograms or plastic wells. In both assay systems the 3'-sulphated Le(a)/Le(x) type tetrasaccharides [formula: see text] were more strongly bound than 3'-sialyl analogues. A considerable binding was observed to the 3'-sulphated oligosaccharide backbone in the absence of fucose but not to a 3'-sialyl analogue or fuco-oligosaccharide analogues lacking sulphate or sialic acid.

Novel sulfated ligands for the cell adhesion molecule E-selectin revealed by the neoglycolipid technology among O-linked oligosaccharides on an ovarian cystadenoma glycoprotein.

E-selectin is the inducible adhesion protein on the surface of endothelial cells which has a crucial role in the initial stages of recruitment of leucocytes to sites of inflammation. In addition, it is almost certainly involved in tumor cell adhesion and metastasis. This report is concerned with identification of a new class of oligosaccharide ligand--sulfate-containing--for the human E-selectin molecule from among oligosaccharides on an ovarian cystadenoma glycoprotein.

Spectrum of sialylated and nonsialylated fuco-oligosaccharides bound by the endothelial-leukocyte adhesion molecule E-selectin. Dependence of the carbohydrate binding activity on E-selectin density.

Carbohydrate recognition by the human endothelial-leukocyte adhesion molecule, E-selectin, has been investigated by binding studies using 3H-labeled Chinese hamster ovary cells expressing different levels of the transfected full-length adhesion molecule and a series of structurally defined oligosaccharides linked to the lipid phosphatidylethanolamine dipalmitoate (neoglycolipids) and synthetic glycolipids chromatographed on silica gel plates or immobilized on plastic wells. Evidence is presented for density-dependent binding of the membrane-associated E-selectin not only to 3'-sialyl-lacto-N-fucopentaose II (3'-S-LNFP-II) and 3'-sialyl-lacto-N-fucopentaose III (3'-S-LNFP-III) which express the sialyl Le(a) and sialyl Le(x) antigens, respectively, but also to the nonsialylated analogue LNFP-II; there is a threshold density of E-selectin required for binding to these sialylated sequences, and binding to the nonsialylated analogue is a property only of cells with the highest density of E-selectin expression. The presence of fucose linked to subterminal rather than to an internal N-acetylglucosamine is shown to be a requirement for E-selectin binding, and although the presence of sialic acid 3-linked to the terminal galactose of the LNFP-II or LNFP-III sequences substantially enhances E-selectin binding, the presence of 6-linked sialic acid abolishes binding.

Specificity of lung surfactant protein SP-A for both the carbohydrate and the lipid moieties of certain neutral glycolipids.

Binding specificity of the major surfactant protein SP-A from human and dog lung has been investigated. Radiobinding experiments have shown that both proteins bind in a Ca(2+)-dependent manner to galactose, mannose, fucose, and glucose linked to bovine serum albumin. These results are in accord with a previous study in which monosaccharides were linked to agarose (Haagsman, H.

Human serum amyloid P is a multispecific adhesive protein whose ligands include 6-phosphorylated mannose and the 3-sulphated saccharides galactose, N-acetylgalactosamine and glucuronic acid.

Carbohydrate recognition by amyloid P component from human serum has been investigated by binding experiments using several glycosaminoglycans, polysaccharides and a series of structurally defined neoglycolipids and natural glycolipids. Two novel classes of carbohydrate ligands have been identified. The first is 6-phosphorylated mannose as found on lysosomal hydrolases, and the second is the 3-sulphated saccharides galactose, N-acetyl-galactosamine and glucuronic acid as found on sulphatide and other acidic glycolipids that occur in neural or kidney tissues or on subpopulations of lymphocytes.

Differential recognition of core and terminal portions of oligosaccharide ligands by carbohydrate-recognition domains of two mannose-binding proteins.

Two different mannose-binding proteins (MBP-A and MBP-C), which show 56% sequence identity, are present in rat serum and liver. It has previously been shown that MBP-A binds to a range of monosaccharide-bovine serum albumin conjugates, and that, among oligosaccharide ligands tested, preferential binding is to terminal nonreducing N-acetylglucosamine residues of complex type N-linked oligosaccharides. In order to compare the binding specificity of MBP-C, an expression system has been developed for production of a fragment of this protein which contains the COOH-terminal carbohydrate-recognition domain.

The spectrum of N-linked oligosaccharide structures detected by enzymic microsequencing on a recombinant soluble CD4 glycoprotein from Chinese hamster ovary cells.

Structures of the N-linked oligosaccharides of a recombinant soluble form of human CD4 glycoprotein (sCD4) have been investigated by enzymic microsequencing. The glycoprotein has two N-glycosylation sites, Asn271 and Asn300, at both of which evidence for the presence of complex type biantennary sialo-oligosaccharides has been obtained previously by mass spectrometric analyses [Carr, S.A.

Response of mononuclear leukocyte cyclic adenosine monophosphate-phosphodiesterase activity to treatment with topical fluorinated steroid ointment in atopic dermatitis.

Our studies of mononuclear leukocyte peripheral blood homogenates demonstrate significantly increased cyclic adenosine monophosphate-specific phosphodiesterase activity in patients with atopic dermatitis who were untreated for 1 week, compared with normal adult nonatopic control subjects. Phosphodiesterase activity is not related to the extent or activity of the patient's disease or the presence or absence of allergic respiratory disease. Enzyme kinetic studies showed a triphasic plot in normal mononuclear leukocytes but a biphasic plot in atopic dermatitis.

The effect of in vitro exposure to histamine on mononuclear leucocyte phosphodiesterase activity in atopic dermatitis.

Increased cyclic AMP (cAMP) phosphodiesterase activity (PDE) is present in the peripheral blood mononuclear leucocytes (MNL) of patients suffering from atopic dermatitis. It is unknown whether this is a primary abnormality or whether it is a consequence of MNL exposure to inflammatory mediators. In this study we have compared the responses of MNL PDE from untreated atopic dermatitis patients, patients on prolonged therapy with topical fluorinated steroids, and normal controls following exposure in vitro to low concentrations of histamine.

Isoenzymes of urinary N-acetyl-beta-D-glucosaminidase (NAG) in patients with renal transplants.

Renal transplant recipients were divided into five categories according to their clinical course from transplantation to their discharge from hospital. Total N-acetyl-beta-D-glucosaminidase (NAG) activity in urine was determined using a chromogenic substrate 2-methoxy-4-(2'-nitrovinyl)-phenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. The isoenzyme composition of the urine of each patient was determined by semi-automated DEAE-cellulose chromatography.

Increased activity of serum amine oxidases in granuloma annulare, necrobiosis lipoidica and diabetes.

Serum monoamine oxidase, diamine oxidase and lysyl oxidase-like activity were measured in patients with granuloma annulare (GA), necrobiosis lipoidica (NL) and diabetes mellitus. In diabetes, all enzyme measurements were raised by a factor of about 2 X 2, and in NL by a factor of about 1 X 5. The rise in patients with GA was small and only significant in the case of benzylamine monoamine oxidase.

Accumulation of sorbitol in endothelial cells--a possible cause of diabetic microangiopathy.

Human umbilical cord endothelial cells accumulate more intracellular sorbitol in response to increased glucose concentration during in vitro incubation. This accumulation of sorbitol by endothelial cells is both concentration and time dependent. Increased concentration of sorbitol in endothelial cells may be important in the pathogenesis of diabetic associated microangiopathy.

Chemotactic peptides modulate adherence of human polymorphonuclear leukocytes to monolayers of cultured endothelial cells.

We have used a new centrifugation assay to examine the effects of highly purified human C5a and C5a des Arg, as well as effects of N-formyl-methionyl-leucyl-phenylalanine (FMLP), on both the extent and strength of human polymorphonuclear leukocyte (PMN) adherence to monolayers of cultured human umbilical vein endothelial cells. At concentrations that were chemotactic for PMN, C5a (0.1 nM), C5a des Arg (5.

Adherence of human polymorphonuclear leukocytes to endothelial monolayers: effects of temperature, divalent cations, and chemotactic factors on the strength of adherence measured with a new centrifugation assay.

Polymorphonuclear leukocytes (PMNs) adhere to endothelial cells at sites of acute inflammation. To examine this phenomenon in vitro, we have developed a new assay to measure adherence of PMNs to cultured endothelial cells. Human PMNs were labeled with 111indium-oxine and incubated in microtiter wells with monolayers of either human umbilical vein or bovine aortic endothelial cells.

Colorimetric assay for N-acetyl-beta-D-glucosaminidase (NAG) in pathological urine using the omega-nitrostyryl substrate: the development of a kit and the comparison of manual procedure with the automated fluorimetric method.

This paper describes a comparison of the recently developed substrate 2-methoxy-4-(2'-nitrovinyl)-phenyl-2-acetamido-2-deoxy-beta-D-gluc opyranoside (MNP-GlcNAc) and the corresponding 4-methylumbelliferyl substrate (4-MU-GlcNAc) for the determination of urinary NAG. A good correlation (r = 0.977) was found between NAG activities in 366 urine samples from renal transplant patients determined by either the fluorimetric method or the colorimetric procedure.

The diagnostic value of urinary enzyme measurements in hypertension.

N-Acetyl-beta-D-glucosaminidase (NAG), beta-D-galactosidase, alkaline phosphatase (ALP) and leucine aminopeptidase (LAP) were assayed in the urine of 100 normal and 112 hypertensive subjects. Age-related urinary activities for these enzymes in the normotensive control subjects are presented. A new procedure for the assay of urinary ALP using 2-methoxy-4-(2'-nitrovinyl)phenyl (MNP) phosphate is described.

Colorimetric assays for N-acetyl-beta-D-glucosaminidase and beta-D-galactosidase in human urine using newly-developed omega-nitrostyryl substrates.

(1) The synthesis of 2-methoxy-4-(2'-nitrovinyl)-phenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (MNP-GlcNAc) and 2-methoxy-4-(2'nitrovinyl)-phenyl beta-D-galactopyranoside (MNP-Gal) as substrates for the assay of NAG and beta-d-galactosidase are described. (2) beta-Glycosidase activities were determined in random urine samples from normal males and females aged between 12 and 87 years and patients with renal disease. (3) Both the MNP N-acetylglucosaminide and MNP galactoside were stable indefinitely, if stored in the solid state at 4 degree C in the dark.

The assay of arlesterase in serum using two new colorimetric substrates, omega-nitrostyrylacetate and propionate.

(1) The synthesis of 4-acetoxy-3-methoxy-omega-nitrostyrene and 4-propionyloxy-3-methoxy-omega-nitrostyrene as substrates for the assay of arylesterase activity is described. These substrates are stable when stored in the dark for at least one year. (2) Serum isolated from normal males and females aged between 25 and 35 years was used as a source of esterase activity.