Identification and characterization of rat AILIM/ICOS, a novel T-cell costimulatory molecule, related to the CD28/CTLA4 family

Activation-inducible lymphocyte immuno-mediatory molecule (AILIM) is an inducible cell surface glycoprotein expressed on thymocytes and activated lymphocytes. Specific monoclonal antibody to rat AILIM induced the cell aggregation of a rat thymoma cell line and ConA-activated splenocytes. In the present study, we identified the primary structure of two species of rat AILIM by expression cloning. We also cloned mouse and human AILIM homologues and the predicted amino acid sequences were identical to those of the inducible costimulator ICOS/CRP-1, which belongs to the CD28/CTLA4 family. Although the human and mouse AILIM/ICOS molecule is localized on T-cells, the major population of AILIM/ICOS-positive cells in rat splenocyte was CD45RA-positive B-cells. The expression level of AILIM/ICOS on T-cells was relatively low; however, its expression was drastically induced by the treatment with PMA plus Ca-ionophore or the engagement of CD3 and these costimulatory molecules. Almost all T-cells exhibited potency as to its expression. Functional analysis of AILIM/ICOS demonstrated that AILIM-mediated costimulation was relatively weak compared to that of human.     

Structural features of the human salivary mucin, MUC7

Human salivary mucin (MUC7) is characterized by a single polypeptide chain of 357 aa. Detailed analysis of the derived MUC7 peptide sequence reveals five distinct regions or domains: (1) an N-terminal basic, histatin-like domain which has a leucine-zipper segment, (2) a moderately glycosylated domain, (3) six heavily glycosylated tandem repeats each consisting of 23 aa, (4) another heavily glycosylated MUC1- and MUC2-like domain, and (5) a C-terminal leucine-zipper segment. Chemical analysis and semi-empirical prediction algorithms for O-glycosylation suggested that 86/105 (83%) Ser/Thr residues were O-glycosylated with the majority located in the tandem repeats. The high (～25%) proline content of MUC7 including 19 diproline segments suggested the presence of polyproline type structures. CD studies of natural and synthetic diproline-rich peptides and glycopeptides indicated that polyproline type structures do play a significant role in the conformational dynamics of MUC7. In addition, crystal structure analysis of a synthetic diproline segment (Boc-Ala-Pro-OBzl) revealed a polyproline type II extended structure. Collectively, the data indicate that the polyproline type II structure, dispersed throughout the tandem repeats, may impart a stiffening of the backbone and could act in consort with the glycosylated segments to keep MUC7 in a semi-rigid, rod shaped conformation resembling a ‘bottle-brush’ model.     

PI3-kinase and MAP-kinase signaling cascades in AILIM/ICOS- and CD28-costimulated T-cells have distinct functions between cell proliferation and IL-10 production

Both AILIM/ICOS and CD28 provide positive costimulatory signals for T-cell activation, resulting in proliferation and cytokine production. In this study, we attempted to clarify the key signaling molecules in T-cell proliferation, and also IL-2 and IL-10 production, during T-cell activation by CD3 induced by costimulation with either AILIM/ICOS or CD28. We examined the role of both the PI3-kinase/Akt pathway and MAP kinase family members such as ERK1/2, JNK, and p38 kinase in this process. PI3-kinase and Erk1/2 were shown to potentially regulate primary T-cell activation and subsequent proliferation via both AILIM/ICOS- or CD28-mediated costimulation and the Erk signaling cascade was essential for this proliferation induction and also for IL-2 production. The JAK inhibitor, AG490, inhibited this induction. Our studies indicate that IL-2 is necessary for induction of T-cell proliferation and that the quantities of IL-2 produced by AILIM/ICOS ligation are also sufficient for T-cells to proliferate. In contrast, inhibition of Akt and p38, that are phosphorylated by both AILIM/ICOS and CD28-ligation, could downregulate IL-10 production but not T-cell proliferation. These data raise the interesting possibility that the signaling cascades between T-cell proliferation and IL-10 production are regulated by different molecules in AILIM/ICOS- and CD28-costimulated T-cells.     

Efficient and systematic synthesis of a small glycoconjugate library having human complex type oligosaccharides

To prepare a small library of homogeneous glycoconjugates with varying oligosaccharide structures, a combinatorial strategy was employed. The target glycopeptide was divided into two peptide segments (A and B) and both were prepared by solid phase peptide synthesis. These peptides, which can be coupled by native chemical ligation through an amide bond, were subsequently coupled to two kinds of human complex type oligosaccharides. This process systematically afforded the desired glycoconjugate library.     

The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria. Localization of haem group and domains in the polypeptide chain

Cytochrome P-450scc consists of two domains linked with a short loop of the polypeptide chain; under hydrolysis by trypsin the domains retain their associated state due to rigid noncovalent interactions. A partial separation of the domains by gel-chromatography on Sephadex G-200 with retention of a haem group in domain I has been achieved after incubation of the trypsin-modified cytochrome P-450scc in 50 mM phosphate buffer (pH 7.2)/1 M NaCl/0.3% sodium cholate/0.3% Tween 80. The separation of domains I and II to individual fragments of the haemoprotein polypeptide chain has been achieved by chromatography under denaturation conditions on the activated thiopropyl-Sepharose via a selective covalent immobilization of domain II. Dissociation of a complex of domains I and II has been effectuated in the presence of 7 M guanidine. Structural characteristics of individual domains have been investigated. It is established that domain I containing a haem group is the N-terminal moiety, and domain II, the C-terminal moiety of the polypeptide chain of cytochrome P-450scc. The pathways of limited trypsinolysis of the native cytochrome P-450scc have been determined. The peptides containing cysteine residues localized on the surface of domain II and responsible for the interaction of haemoprotein with activated thiopropyl-Sepharose have been isolated in a homogeneous form and their amino-acid sequences have been assessed.     

Isolation and sequence analysis of the glycosaminoglycan attachment site of type IX collagen

Type IX collagen from chick embryonic cartilage is unique among the collagens in that it contains chondroitin sulfate covalently linked to the alpha 2(IX) polypeptide chain. We have isolated and sequenced the glycosaminoglycan-containing peptide released by collagenase digestion from type IX collagen, labeled biosynthetically with [35SO4] and 3H-aminoacids. This peptide was purified by gel filtration and, following chondroitinase ABC digestion, by reverse-phase high performance liquid chromatography. The amino acid sequence obtained for this peptide has 23 residues, beginning and ending with a collagenous sequence, indicating that it spans an internal noncollagenous domain. Comparison of this sequence with the one predicted from cDNA clone pYN 1738 for the alpha 1(IX)chain and pYN 1731 and pDM 222 for the alpha 2(IX)chain revealed the peptide to be the noncollagenous NC3 domain of alpha 2(IX). The glycosylated sequence Val-Glu-Gly-Ser*-Ala-Asp- of type IX collagen does not have the Ser-Gly normally functioning as the attachment sequence but does have an acidic residue preceding the serine which should improve the acceptability of this sequence for the xylosyltransferase. That it is an adequate acceptor can be inferred from the observation that type IX collagen carries a glycosaminoglycan chain on over 70% of the molecules isolated.     

Conformation of the third domain of turkey ovomucoid in solution. Structural analysis by two-dimensional Overhauser nuclear effect spectroscopy]

The conformations of a polypeptide chain of turkey ovomucoid third domain and its modified form with split reactive site peptide bond Leu-18--Glu-19 have been determined by the literary two-dimensional nuclear Overhauser effect spectroscopy data using an earlier suggested method. It has been found that the polypeptide domain backbone contains an alpha-helical fragment (residues 32-47), five segments having extended conformation (1-5, 11-17, 19-25, 29-31, 48-50) and beta-turn type 1 (26-29). Segments 23-26, 28-31 and 50-51 form an antiparallel beta-structure. Conformational states of the residues entering irregular domain segments have been analysed. Splitting of the reactive site peptide bond Leu-18--Glu-19 is shown to cause insignificant changes in the conformations of a number of amino acid residues except for Val-6 and Asp-7 ones which undergo essential conformational alterations. The conformations of domain in solution and of japanese quail ovomucoid third domain in crystal have been compared. The root-mean-square deviations for phi and psi angles indicate their high similarity. The conformations of turkey ovomucoid third domain and proteinase inhibitor BUSI IIA in solution have been analysed. In spite of moderate (50%) homology of primary structures, some 75% of amino acid residues are shown to have close conformational phi and psi parameters.     

Sulfated oligosaccharides in human lysosomal enzymes

Cathepsin D, arylsulfatase A and the alpha-chain of beta-hexosaminidase are synthesized in human fibroblasts as sulfated polypeptides. The sulfate is added posttranslationally. Its half-life is less than one-tenth of that of the respective polypeptide chains. The sulfate residues were found on asparagine-linked oligosaccharides sensitive to endoglycosidase F and peptide: N-glycosidase F and resistant to endoglycosidase H. Inhibition of formation of complex type oligosaccharides by 1-deoxy-manno-nojirimycin prevented sulfation, indicating that the sulfate residues were added to complex type oligosaccharides.     

Glycosylation of the two <Emphasis Type="Italic">O</Emphasis>-glycosylated domains of human MUC2 mucin in patients transposed with artificial urinary bladders constructed from proximal colonic tissue

Transposition of intestinal segments is frequently used for bladder reconstruction. Following transposition, bowel segments continue to produce mucus and a correlation between excessive mucus production and complications such as urinary tract infection or catheter blockage has been observed for a long time. However, no information is currently available on the change of mucin expression and glycosylation under these abnormal conditions. In this study, the variable number tandem repeat region and the irregular repeat domain of human MUC2 were isolated as two glycopeptide populations after reduction and trypsin digestion followed by gel chromatography from urine of patients transposed with urinary bladders. After alkaline borohydride treatment, the oligosaccharides released from the whole MUC2 mucin and the two glycosylated domains were investigated by nanoESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem mass spectrometry). More than 60 different glycans were identified, mainly based on sialylated core 3 structures. Some core 1, 2 and 4 oligosaccharides were also found. Most of the structures were acidic with NeuAc residues mainly α2–6 linked to the N-acetylgalactosaminitol and sulphate residues exclusively 3-linked to galactose. No expression of blood group A and B or Sda/Cad determinants was observed. Similar patterns of glycosylation were found in the tandem repeat region and the irregular repeat domain and the level of expression of the major oligosaccharides were in the same order of magnitude. The most interesting feature of this study was that sialyl-Tn antigen, which is considered as a tumour antigen, was the oligosaccharide most highly expressed. This result suggests that mucins from intestinal transposed segments are abnormally glycosylated.     

Structure of the carbohydrate chain of glycosylated porcine prolactin

Cyanogen bromide and chymotryptic fragments both containing a carbohydrate chain were obtained from the glycosylated form of porcine prolactin. By means of the glycosidase hydrolysis and the Smith degradation the carbohydrate chain of the glycoprotein was shown to be of the "complex" type of N-bound oligosaccharides and identical to the chain of the luteinizing hormone.     

Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes in transfected baby hamster kidney cells.

BHK cells transfected with human lysosomal acid phosphatase (LAP) cDNA (CT29) expressed 70-fold higher enzyme activities of acid phosphatase than non-transfected BHK cells. The CT29-LAP was synthesized in BHK cells as a heterogeneously glycosylated precursor that was tightly membrane associated. Transfer to the trans-Golgi was associated with a small increase in size (approximately 7 kd) and partial processing of the oligosaccharides to complex type structures. CT29-LAP was transferred into lysosomes as shown by subcellular fractionation, immunofluorescence and immunoelectron microscopy. Lack of mannose-6-phosphate residues suggested that transport does not involve mannose-6-phosphate receptors. Part of the membrane-associated CT29-LAP was processed to a soluble form. The mechanism that converts CT29-LAP into a soluble form was sensitive to NH4Cl, and reduced the size of the polypeptide by 7 kd. In vitro translation of CT29-derived cRNA in the presence of microsomal membranes yielded a CT29-LAP precursor that is protected from proteinase K except for a small peptide of approximately 2 kd. In combination with the sequence data available for LAP, these observations suggest that CT29-LAP is synthesized and transported to lysosomes as a transmembrane protein. In the lysosomes, CT29-LAP is released from the membrane by proteolytic cleavage, which removes a C-terminal peptide including the transmembrane domain and the cytosolic tail of 18 amino acids.     

Structure of LEP100, a glycoprotein that shuttles between lysosomes and the plasma membrane, deduced from the nucleotide sequence of the encoding cDNA

LEP100, a membrane glycoprotein that has the unique property of shuttling from lysosomes to endosomes to plasma membrane and back, was purified from chicken brain. Its NH2-terminal amino acid sequence was determined, and an oligonucleotide encoding part of this sequence was used to clone the encoding cDNA. The deduced amino acid sequence consists of 414 residues of which the NH2-terminal 18 constitute a signal peptide. The sequence includes 17 sites for N-glycosylation in the NH2-terminal 75% of the polypeptide chain followed by a region lacking N-linked oligosaccharides, a single possible membrane-spanning segment, and a cytoplasmic domain of 11 residues, including three potential phosphorylation sites. Eight cysteine residues are spaced in a regular pattern through the lumenal (extracellular) domain, while a 32-residue sequence rich in proline, serine, and threonine occurs at its midpoint. Expression of the cDNA in mouse L cells resulted in targeting of LEP100 primarily to the mouse lysosomes.     

A structural model of human erythrocyte protein 4.1

Limited proteolysis and specific chemical cleavage methods have enabled a detailed structural characterization of human erythrocyte protein 4.1. This protein is composed of two chemically very similar polypeptide chains (a and b) with apparent molecular masses of 80,000 and 78,000 daltons. Cleavage of protein 4.1 at cysteine residues by 2-nitro-5-thiocyanobenzoic acid produces a series of doublets which differ by approximately 2,000 daltons and have identical peptide maps. Alignment of these peptides by mapping analysis has localized 4 cysteine residues within a 17,000-dalton segment on both a and b polypeptides. Mild chymotryptic treatment at 0 degrees C cleaves protein 4.1 primarily in three central locations and generates two families of unrelated peptides. Analysis of these fragments in two-dimensional gels and by peptide mapping reveals an unusual polarity in protein 4.1 structure in that each polypeptide chain contains two segments, one relatively acidic the other basic, that are segregated at opposite ends of the molecule. The basic region is digested into a cysteine-rich 30,000-dalton domain which resists further breakdown while the acidic region is readily degraded into smaller fragments. The peptides derived from the acidic region all appear as doublets suggesting that protein 4.1 a and b polypeptides differ close to the terminus of the acidic end. Similar phosphorylation sites occur on both polypeptides within a segment some 24,000-34,000 daltons from the acidic terminus.     

Domain structure and 1H-n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.

The pyruvate dehydrogenase complex of Bacillus stearothermophilus was treated with Staphylococcus aureus V8 proteinase, causing cleavage of the dihydrolipoamide acetyltransferase polypeptide chain (apparent Mr 57 000), inhibition of the enzymic activity and disassembly of the complex. Fragments of the dihydrolipoamide acetyltransferase chains with apparent Mr 28 000, which contained the acetyltransferase activity, remained assembled as a particle ascribed the role of an inner core of the complex. The lipoic acid residue of each dihydrolipoamide acetyltransferase chain was found as part of a small but stable domain that, unlike free lipoamide, was able still to function as a substrate for reductive acetylation by pyruvate in the presence of intact enzyme complex or isolated pyruvate dehydrogenase (lipoamide) component. The lipoyl domain was acidic and had an apparent Mr of 6500 (by sedimentation equilibrium), 7800 (by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) and 10 000 and 20 400 (by gel filtration in the presence and in the absence respectively of 6M-guanidinium chloride). 1H-n.m.r. spectroscopy of the dihydrolipoamide acetyltransferase inner core demonstrated that it did not contain the segments of highly mobile polypeptide chain found in the pyruvate dehydrogenase complex. 1H-n.m.r. spectroscopy of the lipoyl domain demonstrated that it had a stable and defined tertiary structure. From these and other experiments, a model of the dihydrolipoamide acetyltransferase chain is proposed in which the small, folded, lipoyl domain comprises the N-terminal region, and the large, folded, core-forming domain that contains the acetyltransferase active site comprises the C-terminal region. These two regions are separated by a third segment of the chain, which includes a substantial region of polypeptide chain that enjoys high conformational mobility and facilitates movement of the lipoyl domain between the various active sites in the enzyme complex.     

Glycosylation and proteolytic processing of 70 kDa C-terminal recombinant polypeptides of Plasmodium falciparum merozoite surface protein 1 expressed in mammalian cells

The cDNAs that encode the 70 kDa C-terminal portion of Plasmodium falciparum merozoite surface protein 1 (MSP-1), with or without an N-terminal signal peptide sequence and C-terminal glycosylphosphatidylinositol (GPI) signal sequence of MSP-1, were expressed in mammalian cell lines via recombinant vaccinia virus. The polypeptides were studied with respect to the nature of glycosylation, localization, and proteolytic processing. The polypeptides derived from the cDNAs that contained the N-terminal signal peptide were modified with N -linked high mannose type structures and low levels of O -linked oligosaccharides, whereas the polypeptides from the cDNAs that lacked the signal peptide were not glycosylated. The GPI anchor moiety is either absent or present at a very low level in the polypeptide expressed from the cDNA that contained both the signal peptide and GPI signal sequences. Together, these data establish that whereas the signal peptide of MSP-1 is functional, the GPI anchor signal is either nonfunctional or poorly functional in mammalian cells. The polypeptides expressed from the cDNAs that contained the signal peptide were proteolytically cleaved at their C-termini, whereas the polypeptides expressed from the cDNAs that lacked the signal peptide were uncleaved. While the polypeptide expressed from the cDNA containing both the signal peptide and GPI anchor signal was truncated by approximately 14 kDa at the C-terminus, the polypeptide derived from the cDNA with only the signal peptide was processed to remove approximately 6 kDa, also from the C-terminus. Furthermore, the polypeptides derived from cDNAs that lacked the signal peptide were exclusively localized intra-cellularly, the polypeptides from cDNAs that contained the signal peptide were predominantly intracellular, with low levels on the cell surface; none of the polypeptides was secreted into the culture medium to a detectable level.These results suggest that N -glycosylation alone is not sufficient for the efficient extracellular transport of the recombinant MSP-1 polypeptides through the secretory pathway in mammalian cells.     

Biosynthesis of intestinal microvillar proteins. The effect of swainsonine on post-translational processing of aminopeptidase N.

The post-translational processing of pig small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in organ-cultured mucosal explants. Exposure of the explants to swainsonine, an inhibitor of Golgi mannosidase II, resulted in the formation of a Mr-160000 polypeptide, still sensitive to endo-beta-N-acetylglucosaminidase H. Swainsonine caused only a moderate inhibition of transport of the enzyme through the Golgi complex and the subsequent expression in the microvillar membrane. This may imply that the trimming of the high-mannose core and complex glycosylation of N-linked oligosaccharides is not essential for the transport of aminopeptidase N to its final destination. A different type of processing was observed to take place in the presence of swainsonine, resulting in a considerable increase in apparent Mr (from 140000 to 160000). This processing could not be ascribed to N-linked glycosylation, since treatment of the Mr-160000 polypeptide with endo-beta-N-acetylglucosaminidase H only decreased its apparent Mr by 15000. The susceptibility of the mature Mr-166000 polypeptide, but not the Mr-140000 polypeptide, to mild alkaline hydrolysis suggests that aminopeptidase N becomes glycosylated with O-linked oligosaccharides during its passage through the Golgi complex. Aminopeptidase N was not labelled by [3H]palmitic acid, indicating that the processing of the enzyme does not include acylation.     

Localization and structure of the asparagine-linked oligosaccharides of type IV collagen from glomerular basement membrane and lens capsule.

Analysis of the Sephacryl S-200 fractionated type IV collagen domains from bovine and human glomerular basement membranes (GBM) and calf anterior lens capsule (ALC) indicated that Asn-linked oligosaccharides are primarily or exclusively localized in the 7 S region, whereas the hydroxylysine-linked Glc alpha 1----2Gal disaccharides (Glc-Gal-Hyl) are present in all the major segments of the molecule (7 S, NC1, and helical domain); no Ser/Thr-linked saccharide were detected. The Asn-linked carbohydrate units observed in the 7 S domain (Mr approximately 300,000) occurred in a number equal to the 12 polypeptide chains constituting this cross-linked region, and this was consistent with lectin blots of the reduced electrophoretically resolved 7 S components. Fractionation of the N-glycanase and endo-beta-N-acetylglucosaminidase-released oligosaccharides by concanavalin A affinity and high performance liquid chromatography indicated that the Asn-linked carbohydrate occurred predominantly in the form of complex tri- and biantennary units, although submolar amounts of polymannose variants (Man5-7GlcNAc2) were also present in calf ALC and bovine GBM. Structural studies of the complex N-linked oligosaccharides employing hydrazine/nitrous acid fragmentation and glycosidase digestions indicated a pattern in which there was complete fucosylation of the innermost GlcNAc residue of the Man3GlcNAc2 core but only sparse substitution with capping groups of the nonrepeating N-acetyllactosamine branches. Whether tri- or biantennary, the oligosaccharides from bovine GBM contained only one capping residue, in the form of either NeuAc or alpha-D-Gal, whereas those from ALC had only a single alpha-D-Gal and no NeuAc; human GBM oligosaccharides were devoid of both NeuAc and alpha-D-Gal. The absence of terminal alpha-D-Gal in the human 7 S domain was reflected in its lack of reactivity with Bandeiraea simplicifolia I and from its failure to yield Gal alpha 1----3Gal beta 1----4 [3H]anhydromannitol after hydrazine/nitrous acid/NaB3H4 treatment. Application of the latter procedure to the collagen domains yielded, in addition to fragments from the N-linked oligosaccharides, a disaccharide (Glc alpha 1----2[3H]galactitol) derived from the Glc-Gal-Hyl units. The localization of Asn-linked carbohydrate units in the evolutionarily conserved 7S domain of type IV collagens suggests that these oligosaccharides may play a role in the assembly of the collagen network of basement membranes.     

Conformational flexibility and folding of synthetic peptides representing an interdomain segment of polypeptide chain in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

Synthetic peptides (32 residues in length) were synthesized with amino acid sequences identical to, or related to, the long (alanine + proline)-rich region of polypeptide chain that links the innermost lipoyl domain to the dihydrolipoamide dehydrogenase-binding domain in the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The 400-MHz 1H NMR spectra of the peptide (Mr approximately 2800) closely resembled the sharp resonances in the spectrum of the intact complex (Mr approximately 5 x 10(6], and the apparent pKa (6.4) of the side chain of a histidine residue in one of the peptides was found to be identical to that previously observed for a histidine residue inserted by site-directed mutagenesis into the corresponding position in the same (alanine + proline)-rich region of a genetically reconstructed enzyme complex. These results strongly support the view that the three long (alanine + proline)-rich regions of the dihydrolipoyl acetyltransferase chains are exposed to solvent and enjoy substantial conformational flexibility in the enzyme complex. More detailed analysis of the peptides by circular dichroism and by 1H and 13C NMR spectroscopy revealed that they were disordered in structure but were not random coils. In particular, all the Ala-Pro peptide bonds were greater than 95% in the trans configuration, consistent with a stiffening of the peptide structure. Differences in the sequences of the three long (alanine + proline)-rich segments may reflect structural tuning of these segments to optimize lipoyl domain movement in enzyme catalysis.     

Isolation and complete amino-acid sequence of the small polypeptide from light-harvesting pigment-protein complex I (B870) of Rhodopseudomonas capsulata

The small bacteriochlorophyll-binding polypeptide of the light-harvesting complex B870 was extracted from the intracytoplasmic membrane of the strain A1a+ of Rhodopseudomonas capsulata with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60 using the same solvent. The polypeptide obtained from the peak fraction III was found to be homogeneous and identical with the small polypeptide isolated from the B870 complex as shown by dodecyl sulfate/polyacrylamide gel electrophoresis, amino acid composition and N-terminal sequence. The complete amino acid sequence is given. The relative molecular mass based on the amino acid sequence is 5341. The polarity of amino acids is 35.42%. The C-terminal part of the peptide chain from residue 29 to 48 is hydrophobic and includes one His residue.