ABSTRACT
The siglecs (sialic acid-binding immunoglobulin-like lectins) mediate sialic acid-dependent cellular interactions and may in some cases signal through SH2-binding domains. In addition to the previously characterized siglecs, sialoadhesin, CD22, CD33 and myelin-associated glycoprotein, several new ones, siglec-5, siglec-7 and siglec-8, have recently been cloned. Although these novel receptors have generated considerable interest as therapeutic targets because of their expression pattern on immune cells, very little is known about how their lectin activity is regulated. Previous studies with sialoadhesin, CD22 and CD33 have shown that siglec glycosylation has significant effects on binding. To determine any differences in the glycan composition of siglec-5, siglec-7 and siglec-8 that may modify their function, we released and characterized the N-linked oligosaccharide distribution in these three glycoproteins. The glycan pools from siglec-5 and siglec-7 contained a larger proportion of sialylated and core-fucosylated biantennary, triantennary and tetra-antennary oligosaccharides, whereas the carbohydrate mixture released from siglec-8 is noticeably less sialylated and is more abundant in 'high-mannose'-type glycans. In addition, we show that, in contrast with CD22 and CD33, mutating the conserved potentially N-linked glycosylation site in the first domain has no effect on binding mediated by siglec-5 or siglec-7.
Subject(s)
Antigens, Differentiation, Myelomonocytic/chemistry , Antigens, Differentiation, Myelomonocytic/metabolism , Asparagine/metabolism , Lectins , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Oligosaccharides/analysis , Amino Acid Motifs , Amino Acid Sequence , Animals , Antigens, CD/chemistry , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, Differentiation, B-Lymphocyte/chemistry , Antigens, Differentiation, B-Lymphocyte/genetics , Antigens, Differentiation, B-Lymphocyte/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , CHO Cells , Carbohydrate Conformation , Carbohydrate Sequence , Cell Line , Cricetinae , Erythrocytes/metabolism , Glycosylation , Humans , Ligands , Mass Spectrometry , Membrane Glycoproteins/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed/genetics , N-Acetylneuraminic Acid/metabolism , Neuraminidase/metabolism , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Protein Binding , Sequence Alignment , alpha-L-Fucosidase/metabolismABSTRACT
A single dose of puromycin aminonucleoside (PAN) given parenterally to rats induces ultrastructural glomerular changes and a nephrotic syndrome similar in many respects to human minimal change nephropathy. The exact aetiologies of both the human and the experimental syndromes are unknown, and are probably multifactorial. However, among the observed consequences in humans and rats is increased plasma protein excretion in urine, beginning in the latter typically 3-6 days after PAN administration. In view of this, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to profile urinary proteins during PAN-induced nephrotoxicity and subsequent recovery in the rat. In addition, urinary high performance liquid chromatography (HPLC) profiles and high resolution proton nuclear magnetic resonance (NMR) spectroscopy has been utilised to simultaneously detect toxin-induced changes in the relative concentrations of a number of metabolites. The proteomic approach, in conjunction with these other techniques, has the potential to provide significantly more mechanistic information than is provided readily by traditional clinical chemistry.
Subject(s)
Kidney Glomerulus/drug effects , Proteome , Puromycin Aminonucleoside/toxicity , Animals , Chromatography, High Pressure Liquid , Electrophoresis, Gel, Two-Dimensional , Kidney Glomerulus/metabolism , Magnetic Resonance Spectroscopy , Male , Proteinuria/metabolism , Rats , Rats, Sprague-Dawley , Spectrometry, Fluorescence , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Rhizopus delemar lipase catalysed ester hydrolysis of the alpha-methoxy-beta-phenylpropanoate 1 affords the (R)-(+) and (S)-(-) isomers in > 84% enantiomeric excess. Absolute stereochemistry was determined by a single crystal X-ray analysis of a related synthetic analogue. The activity of these two enantiomers on glucose transport in vitro and as anti-diabetic agents in vivo is reported and their unexpected equivalence attributed to an enzyme-mediated stereospecific isomerisation of the (R)-(+) isomer. Binding studies using recombinant human PPARgamma (peroxisomal proliferator activated receptor gamma), now established as a molecular target for this compound class, indicate a 20-fold higher binding affinity for the (S) antipode relative to the (R) antipode.
Subject(s)
Hypoglycemic Agents/chemical synthesis , Hypoglycemic Agents/pharmacology , Phenylpropionates/chemical synthesis , Animals , Crystallography, X-Ray , Models, Chemical , Models, Molecular , Rats , Receptors, Cytoplasmic and Nuclear/metabolism , Stereoisomerism , Time Factors , Transcription Factors/metabolismABSTRACT
Studies are reported on the effect of the sodium salt of phytic acid on the resolution of peptides and proteins. Improved separation in the case of peptides is shown to be due to ion-ion pairing interactions between the positively charged peptides and the phytic acid polyanionic species. The improved peak shapes related to the proteins can be interpreted in terms of the sample preconcentration due to injection of analytes from a water medium to one of high ionic strength.
