The small Gtpase ras is involved in growth factor-regulated expression of the alpha1 integrin subunit in PC12 cells.

PC12 cells interact with several growth factors (e. g. EGF, FGF, and NGF) via specific tyrosine receptor kinases, resulting in cell proliferation or neuronal differentiation. The small GTPase Ras is known to be involved in downstream signaling of these growth factor receptors. Furthermore, cell-matrix interactions mediated by integrins, as well as integrin-induced signaling, are also involved in growth factor-stimulated signal transduction in PC12 cells. In this study we determined the expression of the alpha1 integrin subunit in response to EGF and NGF in PC12 wild-type (wt) cells, and in PC12 cells overexpressing an inactive H-Ras protein (RasN17). In PC12 wt cells, alpha1 integrin expression is upregulated by EGF and NGF. Cell surface expression of alpha1beta1integrin is also enhanced in growth factor-treated cells. This upregulation leads to increased alpha1beta1-specific adhesion to collagen. In cells expressing the dominant-negative RasN17 variant, alpha1 integrin expression and alpha1beta1-specific adhesion remain unchanged in response to both growth factors.

Evidence for efficient uptake and incorporation of sialic acid by eukaryotic cells.

Sialic acids are the most abundant terminal carbohydrate moiety on cell surface glycoconjugates in eukaryotic cells and are of functional importance for many biological ligand-receptor interactions. It is a widely accepted view that sialic acids cannot be efficiently taken up from the extracellular space by eukaryotic cells. To test this assumption, we cultivated two recently identified human hematopoetic cell lines which are hyposialylated due to a deficiency in de novo sialic acid biosynthesis in the presence of N-acetylneuraminic acid (NeuAc), the most frequently found sialic acid. Surprisingly, NeuAc medium supplementation rapidly and potently compensated for the endogenous hyposialylation in a concentration-dependent manner, resulting in the presentation of cell surface sialoglycans involved in cell adhesion, virus infection and signal transduction. We provide several lines of experimental evidence that all suggest that NeuAc was neither extracellularly incorporated nor degraded to a less complex sugar before uptake. Importantly, NeuAc induced a marked increase in intracellular CMP-NeuAc levels in both human cell lines and in primary cells regardless of the prior sialylation status of the cells. Studies employing 9-[3H]NeuAc revealed an uptake consistent with the observed incorporation of unlabeled NeuAc. We propose the existence of an efficient uptake mechanism for NeuAc in eukaryotic cells.

Efficient biochemical engineering of cellular sialic acids using an unphysiological sialic acid precursor in cells lacking UDP-N-acetylglucosamine 2-epimerase.

Sialic acids comprise a family of terminal sugars essential for a variety of biological recognition systems. N-Propanoylmannosamine, an unphysiological sialic acid precursor, is taken up and metabolized by mammalian cells resulting in oligosaccharide-bound N-propanoylneuraminic acid. N-Propanoylmannosamine, applied to endogenously hyposialylated subclones of the myeloid leukemia HL60 and of the B-cell lymphoma BJA-B, both deficient in UDP-N-acetylglucosamine 2-epimerase, is efficiently metabolized to CMP-N-propanoylneuraminic acid resulting in up to 85% of glycoconjugate-associated sialic acids being unphysiological N-propanoylneuraminic acid. Thus, UDP-N-acetylglucosamine 2-epimerase-deficient cell lines provide an important experimental progress in engineering cells to display an almost homogeneous population of defined, structurally altered sialic acids.

Eating disorders: a role for dipeptidyl peptidase IV in nutritional control.

Dipeptidyl peptidase IV (DPP IV), a serine protease with broad tissue distribution and known activity in serum, has been postulated to modulate nutrition control by modification or inactivation of peptide hormones operating in the enteroinsular axis. We hypothesized that changes of DPP IV activity in serum are related to the nutrition status of patients with eating disorders. Serum DPP IV activity was measured in 52 patients (28 with anorexia nervosa and 24 with bulimia nervosa) in four consecutive weekly analyses. Simultaneously, the number of CD26 (DPP IV)-positive peripheral blood lymphocytes was counted. The same analyses were carried out in 28 healthy female volunteers. In week 1 and throughout the observation period, DPP IV activity in the sera of patients with anorexia nervosa and, to a lesser extent, those with bulimia nervosa was elevated in comparison to that of healthy controls (week 1: means = 92.8 U/L for anorexia-nervosa patients and 89.3 U/L for bulimia-nervosa patients versus 74.7 U/L for healthy control subjects, P = 0.014; weeks 1-4: 91.8 U/L for anorexia-nervosa patients and 86.2 U/L for bulimia-nervosa patients versus 77.6 U/L for healthy controls, P < 0.001). We assume that the increase in DPP IV serum activity will increase the turnover of distinct peptide hormones with known effects on nutrition control and susceptibility to degradation by DPP IV. The potential impact of an increase in DPP IV activity in serum on satiety and nutrition control contributes to previously reported implications for immune function.

alpha1 Integrin cytoplasmic domain is involved in focal adhesion formation via association with intracellular proteins.

Integrins are heterodimeric adhesion receptors consisting of alpha- and beta-subunits capable of binding extracellular matrix molecules as well as other adhesion receptors on neighbouring cells. These interactions induce various signal transduction pathways in many cell types, leading to cytoskeletal reorganization, phosphorylation and induction of gene expression. Integrin ligation leads to cytoplasmic protein-protein interactions requiring both integrin cytoplasmic domains, and these domains are initiation points for focal adhesion formation and subsequent signal transduction cascades. In previous studies we have shown that the very short cytoplasmic alpha1 tail is required for post-ligand events, such as cell spreading as well as actin stress-fibre formation. In the present paper we report that cells lacking the cytoplasmic domain of the alpha1 integrin subunit are unable to form proper focal adhesions and that phosphorylation on tyrosine residues of focal adhesion components is reduced on alpha1beta1-specific substrates. The alpha1 cytoplasmic sequence is a specific recognition site for focal adhesion components like paxillin, talin, alpha-actinin and pp125FAK. It seems to account for alpha1-specific signalling, since when peptides that mimic the cytoplasmic domain of alpha1 are transferred into cells, they influence alpha1beta1-specific adhesion, presumably by competing for binding partners. For alpha1 integrin/protein binding, the conserved Lys-Ile-Gly-Phe-Phe-Lys-Arg motif and, in particular, the two lysine residues, are important.

Biochemical engineering of the side chain of sialic acids increases the biological stability of the highly sialylated cell adhesion molecule CEACAM1.

The biological half-life time of many glycoproteins is regulated via terminal sialic acids. In this study we determined the half-lives of two different cell adhesion molecules, CEACAM1 and the alpha1-integrin subunit, in PC12-cells before and after biochemical engineering the side chain of sialic acids by the use of N-propanoylmannosamine. Both are transmembrane glycoproteins. While the immunoglobulin superfamily member CEACAM1 mediates homophilic cell-cell adhesion the alpha1-integrin subunit is involved in cell-matrix interactions. We found that the half-life of the highly sialylated CEACAM1 is increased from 26 to 40 h by replacement of the N-acetylneuraminic acid by the novel, engineered N-propanoylneuraminic acids, whereas the half-life of the alpha1-integrin subunit remains unaffected under the same conditions. This demonstrates that biochemical engineering not only modulates the structure of cell surface sialic acids, but that biochemical engineering also influences biological stability of defined glycoproteins.

Biosynthesis of N-acetylneuraminic acid in cells lacking UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.

The first two steps in mammalian biosynthesis of N-acetylneuraminic acid, an important carbohydrate moiety in biological recognition systems, are performed by the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. A subclone of the human B lymphoma cell line BJA-B K20, lacking UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase mRNA as well as epimerase activity, displayed hyposialylated, functionally impaired cell surface glycoconjugates. Here we show that this cell line surprisingly still retains N-acetylmannosamine kinase activity. A gel filtration analysis of BJA-B K88 control cells, which express UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, revealed two N-acetylmannosamine kinase activity peaks, one co-eluting with UDP-N-acetylglucosamine 2-epimerase activity and one co-eluting with N-acetylglucosamine kinase. For this enzyme previous studies already showed a ManNAc kinase activity in vitro. In contrast, the hyposialylated BJA-B K20 subclone displayed only the N-acetylmannosamine kinase peak, co-migrating with N-acetylglucosamine kinase. The CMP-N-acetylneuraminic acid content of both K88 and K20 cells and the sialylation of cell surface glycoconjugates of K20 cells could be significantly increased by supplementing the medium with N-acetylmannosamine. This N-acetylmannosamine-induced increase was drastically reduced by co-supplementation with N-acetylglucosamine only in K20 cells. We therefore propose the phosphorylation of N-acetylmannosamine as a hitherto unrecognized role of N-acetylglucosamine kinase in living cells.

Inhibition of dipeptidyl peptidase IV (DP IV, CD26) activity abrogates stress-induced, cytokine-mediated murine abortions.

In the CBA x DBA/2 mouse model, stress-triggered abortions are mediated by a Th1-like cytokine response of decidual lymphocytes. The factors that determine the cytokine pattern leading to abortion are currently unknown. Dipeptidyl Peptidase IV (DP IV) enhances Th1-cytokine responses and impairs the evolvement of a Th2 cytokine profile. The T-cell-activation antigen, CD26, possesses DP IV activity. The aim of the present study was to investigate the role of DP IV activity and CD26-positive decidual lymphocytes in murine stress-triggered abortions by inhibition of DP IV activity. DBA/2-mated CBA mice were stressed on day 5.5 of pregnancy and received daily injections of an inhibitor of DP IV activity, Ile-thiazolidide (20 micromol/kg). On day 13 of gestation, the animals were sacrificed and the percentage of implants and abortions documented. CD26-positive lymphocytes in spleen and uterine decidua and the intracellular cytokines interferon (IFN)-gamma and interleukin (IL)-10 were determined by flow cytometry. Stressed and nonstressed animals receiving an inactive stereoisomeric form were used as controls. In mice receiving the DP IV inhibitor, stress failed to boost the abortion rate (37.2% versus 13.6%, P < 0.01). IFN-gamma producing cells were increased in stressed animals, but returned to the baseline upon the inhibition of DP IV. The number of IL-10 producing cells was reduced in stressed animals, independent from DP IV inhibition.

Biochemical engineering of the N-acyl side chain of sialic acid: biological implications.

N-Acetylneuraminic acid is the most prominent sialic acid in eukaryotes. The structural diversity of sialic acid is exploited by viruses, bacteria, and toxins and by the sialoglycoproteins and sialoglycolipids involved in cell-cell recognition in their highly specific recognition and binding to cellular receptors. The physiological precursor of all sialic acids is N-acetyl D-mannosamine (ManNAc). By recent findings it could be shown that synthetic N-acyl-modified D-mannosamines can be taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acids in vitro and in vivo. Successfully employed D-mannosamines with modified N-acyl side chains include N-propanoyl- (ManNProp), N-butanoyl- (ManNBut)-, N-pentanoyl- (ManNPent), N-hexanoyl- (ManNHex), N-crotonoyl- (ManNCrot), N-levulinoyl- (ManNLev), N-glycolyl- (ManNGc), and N-azidoacetyl D-mannosamine (ManNAc-azido). All of these compounds are metabolized by the promiscuous sialic acid biosynthetic pathway and are incorporated into cell surface sialoglycoconjugates replacing in a cell type-specific manner 10-85% of normal sialic acids. Application of these compounds to different biological systems has revealed important and unexpected functions of the N-acyl side chain of sialic acids, including its crucial role for the interaction of different viruses with their sialylated host cell receptors. Also, treatment with ManNProp, which contains only one additional methylene group compared to the physiological precursor ManNAc, induced proliferation of astrocytes, microglia, and peripheral T-lymphocytes. Unique, chemically reactive ketone and azido groups can be introduced biosynthetically into cell surface sialoglycans using N-acyl-modified sialic acid precursors, a process offering a variety of applications including the generation of artificial cellular receptors for viral gene delivery. This group of novel sialic acid precursors enabled studies on sialic acid modifications on the surface of living cells and has improved our understanding of carbohydrate receptors in their native environment. The biochemical engineering of the side chain of sialic acid offers new tools to study its biological relevance and to exploit it as a tag for therapeutic and diagnostic applications.

Overexpression of alpha1beta1 integrin directly affects rat mesangial cell behavior.

BACKGROUND: Glomerular mesangial cell (MC) proliferation, hypertrophy, and abnormal matrix remodeling characterized by increased expression of fibronectin, laminin and collagen type IV, and neoexpression of collagen I and III are the main biological features of progressive glomerulonephritis (GN). Especially, persistent pathological matrix remodeling may lead to glomerular scar formation (glomerular scarring). We reported recently that alpha1beta1 integrin, a major collagen receptor for MCs, may be a potential adhesion molecule for MC-mediated pathological collagen matrix remodeling in GN. METHODS: To address further the direct role of alpha1beta1 integrin in MC behavior, such as cell growth and matrix remodeling, alpha1beta1 integrin was overexpressed in MCs by transfecting an expression vector containing a full-length rat alpha1 integrin cDNA. Flow cytometry and immunoprecipitation analysis were applied for selection of transfectants with a stable expression of the alpha1 integrin subunit. The effect of alpha1beta1 integrin overexpression on MC biology was examined with a 3H-thymidine incorporation assay, flow cytometric analysis of cell size and DNA content, Western blot analysis of a cyclin-dependent-kinase inhibitor, p27Kip1, alpha-smooth muscle actin expression, and a collagen gel contraction assay. RESULTS: The alpha1 transfectants displayed a dramatic inhibition of 3H-thymidine incorporation as compared with the mock transfectants. Increased expression of the alpha1 subunit inversely correlated with cell cycle progression and paralleled the expression of p27Kip1 and alpha-smooth muscle actin, as well as the cell size in MCs. In addition, the alpha1-transfectants were able to enhance collagen matrix reorganization effectively. CONCLUSION: These results indicate that MC-alpha1beta1 integrin expression is a critical determinant of MC phenotypes, including cell growth, cell size, and collagen matrix remodeling ability, and thereby contributes to scar matrix remodeling (sclerosis) in GN.

Incorporation of N-propanoylneuraminic acid leads to calcium oscillations in oligodendrocytes upon the application of GABA.

Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis. It has been shown that unnatural sialylation within glial cell cultures can have distinct effects on their proliferation and antigenic profiles. These cultures metabolize N-propanoylmannosamine (N-propanoylneuraminic acid precursor=P-NAP), a synthetic non-physiological precursor of neuraminic acid, resulting in the expression of N-propanoylneuraminic acid in glycoconjugates of their cell membranes [Schmidt, C., Stehling, P., Schnitzer, J., Reutter, W. and Horstkorte, R. (1998) J. Biol. Chem. 273, 19146-19152]. To determine whether these biochemically engineered sialic acids influence calcium concentrations in cells of the oligodendrocyte lineage, mixed glial cultures of oligodendrocytes growing on top of an astrocyte monolayer were exposed to glutamate, histamine, adrenaline, gamma-aminobutyric acid (GABA), high potassium (high K(+)) and ATP. Calcium responses in P-NAP-treated oligodendrocytes were determined by confocal microscopy with the calcium indicator fluo-3 AM, and compared with control cultures. We showed that P-NAP differentially modulated the calcium responses of individual oligodendrocytes when GABA was applied. GABA induced calcium oscillations with up to four spikes per min in 60% of oligodendrocytes when treated with P-NAP.

A guardian angel: the involvement of dipeptidyl peptidase IV in psychoneuroendocrine function, nutrition and immune defence.

Dipeptidyl peptidase IV (DPP IV, also known as CD26; EC 3.4.14.5) is a non-integrin receptor glycoprotein with multiple functions, including cell adhesion, cellular trafficking through the extracellular matrix and co-stimulatory potential during T cell activation. By virtue of its exopeptidase activity, DPP IV plays a key regulatory role in the metabolism of peptide hormones. Based on data emerging from different biomedical specialties, it appears worthwhile to highlight the different facets of DPP IV in nutrition, immune responses and peptide hormone metabolism. The presentation of the complex regulatory circuits in which DPP IV appears to be involved may also serve as a note of caution, in view of attempts to apply selective inhibitors of DPP IV enzymic activity for the treatment of disease, e.g. Type II diabetes.

Roles of cysteines in rat dipeptidyl peptidase IV/CD26 in processing and proteolytic activity.

The multifunctional type II transmembrane glycoprotein, dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), is expressed by almost all mammalian cells and is identical to the adenosine deaminase binding protein CD26 on lymphocytes. The extracellular part of rat DPPIV can be divided into three domains the middle part of which harbors 10 of the 12 highly conserved cysteine residues. The cysteine-rich domain is responsible for DPPIV-binding to collagen I and to extracellular ADA. The participation of distinct cysteines in disulfide bridges is not yet known. Titration experiments have shown the presence of six free cysteines and three disulfide bridges in native rat DPPIV. To investigate the role of distinct cysteines in the structure-function relationships of rat DPPIV we constructed 12 different cysteine point mutations (C299, C326, C383, C455, C650 mutated to G; C337, C395, C445, C448, C473, C552, C763 mutated to S). Intracellular translocation to the cell surface of stable transfected Chinese hamster ovary cells was examined with antibodies against different epitopes of DPPIV. Surface expression of mutants C326G, C445S and C448S is inhibited totally; mutants C337S, C455G, C473S and C552S show weak expression only. In parallel, the half-life of these mutants is reduced to < 10% compared with wild-type enzyme. We were able to show that the specific peptidase activity of the mutant protein depends on cell-surface expression, dimerization and the existence of a 150-kDa form demonstrable by nondenaturing SDS/PAGE. We conclude that cysteine residues 326, 337, 445, 448, 455, 473 and 552 in rat DPPIV are essential for the correct folding and intracellular trafficking of this glycoprotein, and therefore for its normal biological properties.

Dipeptidyl peptidase IV (DPP IV, CD26) in patients with mental eating disorders.

The notion that patients with eating disorders maintain a functional immunosurveillance in spite of severe malnutrition has attracted researchers for years. Dipeptidyl Peptidase IV (DPP IV), a serine protease with broad tissue distribution and known activity in serum, operates in the cascade of immune responses. Membrane-bound DPP IV expressed on lymphocytes, also known as the leukocyte antigen CD26, is considered to participate in T cell activation. We hypothesized that the activity of DPP IV in serum and expression of CD26 in lymphocytes may be altered in patients with eating disorders. Serum DPP IV activity and the number of CD26 (DPP IV)-positive peripheral blood lymphocytes were measured in 44 patients (anorexia nervosa (AN): n = 21, bulimia (B): n = 23) in four consecutive weekly analyses. The analysis of CD26-positive cells included the characterization of CD26-bright and CD26-dim positive subsets. Additionally, the expression of CD25 (IL-2 Receptor alpha chain) was evaluated to estimate the degree of T cell activation. The same analyses were carried out in healthy female volunteers (HC, n = 20). CD26-positive cells were reduced in patients as compared to healthy controls (mean 40.2% (AN) and 41.1% (B) vs. 47.4% (HC), p < 0.01), while the DPP IV activity in serum was elevated (mean 108.4 U/l (AN) and 91.1 U/l (B) vs. 80.3 U/l (HC), p < 0.01). The potential implications of changes in DPP IV expression and serum activity on--and beyond--immune function are discussed.

Molecular cloning and characterization of murine and human N-acetylglucosamine kinase.

N-Acetylglucosamine is produced by the endogenous degradation of glycoconjugates and by the degradation of dietary glycoconjugates by glycosidases. It enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase. In this study we report the isolation and characterization of a cDNA clone encoding the murine enzyme. An open reading frame of 1029 base pairs encodes 343 amino acids with a predicted molecular mass of 37.3 kDa. The deduced amino-acid sequence contains matches of the sequences of eight peptides derived from tryptic cleavage of rat N-acetylglucosamine kinase. The recombinant murine enzyme was functionally expressed in Escherichia coli BL21 cells, where it displays N-acetylglucosamine kinase activity as well as N-acetylmannosamine kinase activity. The complete cDNA sequence of human N-acetylglucosamine kinase was derived from the nucleotide sequences of several expressed sequence tags. An open reading frame of 1032 base pairs encodes 344 amino acids and a protein with a predicted molecular mass of 37.4 kDa. Similarities between human and murine N-acetylglucosamine kinase were 86.6% on the nucleotide level and 91.6% on the amino-acid level. Amino-acid sequences of murine and human N-acetylglucosamine kinase show sequence similarities to other sugar kinases, and all five sequence motifs necessary for the binding of ATP by sugar kinases are present. Tissue distribution of murine N-acetylglucosamine kinase revealed an ubiquitous occurrence of the enzyme and a very high expression in testis. The size of the murine mRNA was 1.35 kb in all tissues investigated, with the exception of testis, where it was 1.45 kb mRNA of the murine enzyme was continuously expressed during mouse development. mRNA of the human enzyme was expressed in all investigated human tissues, as well as in cancer cell lines. In both the tissues and the cancer cell lines, the human mRNA was 1.35 kb in size.

Structure-dependent effects of glucose-containing analogs of platelet activating factor (PAF) on membrane integrity.

Synthetic choline-containing phospholipids comprise a new class of compounds with antineoplastic properties. We have investigated the effect of recently synthesized glucose-containing analogs of lysophosphatidylcholine (glyceroglucophospholipid, Glc-PC) and of lysoplatelet activating factor (Glc-PAF) and its C16, C14 and C12 derivatives (ET-16, ET-14, and ET-12) on proliferation of immortalized human keratinocyte (HaCaT) cells. The data were compared to the ability of the compounds to intercalate into phosphatidylserine liposomes and to form lesions in planar bilayer membranes. A correlation between bioactivity and membrane activity was found. The number of molecules that intercalated into phosphatidylserine liposomes depended on the chemical structure of the compounds and was in the order Glc-PAF approximately ET-16 approximately ET-14 > Glc-PC > ET-12. All compounds induced membrane lesions, and the lesion forming activity was in the same order. Similar activity rankings were found for the release of lactate dehydrogenase from HaCaT cells as a measure of lytic activity and for the influence on cell number as a measure of proliferation. In the latter test, however, proliferation was already inhibited at non-toxic concentrations. From these findings, it may be concluded that the intercalation of the compounds at toxic concentrations leads to the formation of membrane lesions and finally results in membrane rupture leading to cell death.

Sialoforms of dipeptidylpeptidase IV from rat kidney and liver.

Dipeptidylpeptidase IV (DPP IV, CD26), a serine-type exo- and endopeptidase found in the cell surface membrane of many tissues, was employed as a model membrane glycoprotein to study the expression of sialoforms on cell surface glycoproteins. Native, enzymatically active DPP IV was purified from plasma membranes of kidney and liver by lectin affinity chromatography in conjunction with crown ether anion exchange chromatography. The enzyme was gradient-eluted in continuous fractions, all showing a single polypeptide band of about 100 kDa when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing, denaturing conditions. Analysis of the purified DPP IV by isoelectric focusing (IEF) showed that it consists of several polypeptides of different isoelectric points (IP) ranging from 5.5 to 7.0. In vitro- desialylation of the enzyme and subsequent isoelectric focusing revealed that the differences in isoelectric points were due to differences in the degree of sialylation. Differences in the degree of sialylation between the fractions were also demonstrated by SDS-PAGE under nonreducing and nondenaturing conditions. Increased sialylation of the enzyme as demonstrated by isoelectric focusing resulted in increased migration velocity in nonreducing and nondenaturing SDS-polyacrylamide gels. In vitro -desialylation of the enzyme and its resialylation confirmed that sialylation was responsible for this extraordinary migration behavior. The native enzyme was predominantly sialylated via alpha 2, 6-linkage, as shown by lectin affinity blotting employing Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). These findings demonstrate that a distinct membrane glycoprotein may exist in various sialoforms, distinguished from each other by a different number of sialic acid residues. Moreover, these sialoforms can be individually purified by crown ether anion exchange chromatography.

Alterations in expression and in serum activity of dipeptidyl peptidase IV (DPP IV, CD26) in patients with hyporectic eating disorders.

The notion that patients with eating disorders maintain a functional immunosurveillance in spite of severe malnutrition has attracted researchers for years. Dipeptidyl peptidase IV (DPP IV), a serine protease with broad tissue distribution and known activity in serum, operates in the cascade of immune responses. Membrane-bound DPP IV expressed on lymphocytes, also known as the leucocyte antigen CD26, is considered to participate in T-cell activation. We hypothesized that the activity of DPP IV in serum and expression of CD26 in lymphocytes may be altered in patients with eating disorders. Serum DPP IV activity and the number of CD26 (DPP IV)-positive peripheral blood lymphocytes were measured in 34 patients [anorexia nervosa (AN): n = 11, bulimia (B): n = 23] in four consecutive weekly analyses. In addition, the expression of CD25 (interleukin-2 receptor alpha chain) was evaluated to estimate the degree of T-cell activation. The same analyses were carried out in healthy female volunteers (HC, n = 20). CD2-CD26-positive cells were reduced in patients compared with healthy controls [mean 40.2% (AN) and 41.1% (B) versus 47.4% (HC), P < 0.01], while the DPP IV activity in serum was elevated [mean 108.4 U/l (AN) versus 91.1 U/l (B) and 80.3 U/l (HC), P < 0.01]. The potential implications of our observations on, and beyond, immune function are discussed.

In vivo modulation of the acidic N-glycans from rat liver dipeptidyl peptidase IV by N-propanoyl-D-mannosamine.

Derivatives of N-acyl-D-mannosamine differing in the N-acyl-side chain can be metabolically converted into neuraminic acids with corresponding N-acyl side chains. In the present study we show the in vivo modulation of sialic acids in membrane-bound dipeptidyl peptidase IV (CD 26) from rat liver after administration of N-propanoyl-D-mannosamine. Treatment of rats with this unphysiological precursor resulted in an incorporation of N-propanoylneuraminic acid into N-linked glycans of dipeptidyl peptidase IV.

N-glycosylation of the carcinoembryonic antigen related cell adhesion molecule, C-CAM, from rat liver: detection of oversialylated bi- and triantennary structures.

Rat C-CAM is a ubiquitous, transmembrane and carcinoembryonic antigen related cell adhesion molecule. The human counterpart is known as biliary glycoprotein (BGP) or CD66a. It is involved in different cellular functions ranging from intercellular adhesion, microbial receptor activity, signaling and tumor suppression. In the present study N-glycosylation of C-CAM immunopurified from rat liver was analyzed in detail. The primary sequence of rat C-CAM contains 15 potential N-glycosylation sites. The N-glycans were enzymatically released from glycopeptides, fluorescently labeled with 2-aminobenzamide, and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by enzymatic sequencing and MALDI-TOF-MS. Mainly bi- and triantennary complex structures were identified. The presence of type I and type II chains in the antennae of these glycans results in heterogeneous glycosylation of C-CAM. Sialylation of the sugars was found to be unusual; bi- and triantennary glycans contained three and four sialic acid residues, respectively, and this linkage seemed to be restricted to the type I chain in the antennae. Approximately 20% of the detected sugars contain these unusual numbers of sialic acids. C-CAM is the first transmembrane protein found to be oversialylated.