A conserved region in alpha-macroglobulins participates in binding to the mammalian alpha-macroglobulin receptor

Efforts to characterize the receptor recognition domain of alpha-macroglobulins have primarily focused on human alpha 2-macroglobulin (alpha 2M). In the present work, the structure and function of the alpha-macroglobulin receptor recognition site were investigated by amino acid sequence analysis, plasma clearance, and cell binding studies using several nonhuman alpha-macroglobulins: bovine alpha 2M, rat alpha 1-macroglobulin (alpha 1M), rat alpha 1-inhibitor 3 (alpha 1I3), and proteolytic fragments derived from these proteins. Each alpha-macroglobulin bound to the murine peritoneal macrophage alpha-macroglobulin receptor with comparable affinity (Kd approximately 1 nM). A carboxyl-terminal 20-kDa fragment was isolated from each of these proteins, and this fragment bound to alpha-macroglobulin receptors with Kd values ranging from 10 to 125 nM. The amino acid identity between the homologous carboxyl-terminal 20-kDa fragments of human and bovine alpha 2M was approximately 90%, while the overall sequence homology between all carboxyl-terminal fragments studied was 75%. The interchain disulfide bond present in the human alpha 2M carboxyl-terminal 20-kDa fragment was conserved in bovine alpha 2M and rat alpha 1I3, but not in rat alpha 1M. The clearance of each intact alpha-macroglobulin-proteinase complex was significantly retarded following treatment with cis-dichlorodiammineplatinum(II) (cis-DDP). cis-DDP treatment, however, did not affect receptor recognition of purified carboxyl-terminal 20-kDa fragments of these alpha-macroglobulins. A carboxyl-terminal 40-kDa subunit, which can be isolated from rat alpha 1M, bound to the murine alpha-macroglobulin receptor with a Kd of 5 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clearance of desialylated mouse alpha-macroglobulin and murinoglobulin in the mouse.

Mouse alpha-macroglobulin and murinoglobulin were labeled with 125I and utilized for plasma clearance studies performed with mice. Desialylated murinoglobulin was rapidly cleared from the circulation with a half-life of about 5 min. On the other hand, desialylated alpha-macroglobulin showed a biphasic curve: about half was cleared at a rate similar to that of the intact molecule while the remaining half had a shorter half-life of about 20 min which was prolonged by a simultaneous injection of a 200-fold excess of unlabeled asialoorosomucoid. Virtually no cross competition was observed between these asialoglobulins and formaldehyde-treated bovine serum albumin or trypsin-bound alpha-macroglobulin. These results suggest that the intravascular elimination of desialylated alpha-macroglobulin and murinoglobulin is independent of the clearance systems responsible for formaldehyde-modified proteins or proteinase-bound alpha-macroglobulins, and that the structure or spatial arrangement, or both, of oligosaccharide units of alpha-macroglobulin is somewhat different from that of murinoglobulin, resulting in a difference of avidity of interaction with the asialoglycoprotein receptor. The desialylated alpha-macroglobulin and murinoglobulin accumulated principally in the liver.     

Immunocytochemical localization of mutant low density lipoprotein receptors that fail to reach the Golgi complex

In the low density lipoprotein (LDL) receptor system, blocks in intracellular movement of a cell surface receptor result from naturally occurring mutations. These mutations occur in patients with familial hypercholesterolemia. One class of mutant LDL receptor genes (class 2 mutations) produces a receptor that is synthesized and glycosylated in the endoplasmic reticulum (ER) but does not reach the cell surface. These receptors contain serine/threonine-linked (O-linked) carbohydrate chains with core N-acetylgalactosamine residues and asparagine-linked (N-linked) carbohydrate chains of the high mannose type that are only partially trimmed. To determine the site of blockage in transport, we used electron microscope immunohistochemistry to compare the intracellular location of LDL receptors in normal human fibroblasts with their location in class 2 mutant fibroblasts. In normal cells, LDL receptors were located in coated pits, coated vesicles, endosomes, multivesicular bodies, and portions of the Golgi complex. In contrast, the mutant receptors in class 2 cells were almost entirely confined to rough ER and irregular extensions of the rough ER. Metabolic labeling studies with [3H]glucosamine confirmed that these mutant receptors contain core O-linked sugars, suggesting that the enzymes that attach these residues are located in the rough ER or the transitional zone of the ER. These studies establish that naturally occurring mutations in cell surface receptors can cause the receptors to remain trapped in the ER, thereby preventing their normal function and producing a genetic disease.     

Differential binding properties of human pregnancy zone protein- and alpha2-macroglobulin-proteinase complexes to low-density lipoprotein receptor-related protein.

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to alpha2-macroglobulin (alpha2-M). Both alpha-macroglobulins (alpha-Ms) covalently bind proteinases, which is accompanied by the exposure of carboxy terminal receptor recognition domains important for the rapid clearance from the circulation and tissues. It is accepted that the molecule responsible for the clearance of alpha2-M- and PZP-proteinase complexes is the low-density lipoprotein receptor-related protein (LRP). Although both alpha-M-proteinase complexes bind to the same receptor, differences in the binding properties have been reported. In addition, although it is known that the binding of alpha2-M-proteinase complexes to LRP can be blocked by Ni2+, the effect on PZP-proteinase has never been examined. In order to investigate differences in the binding properties of both alpha-Ms to the receptor, we purified LRP from human placenta by affinity chromatography and then analyzed the specificity and affinity of binding of alpha2-M- and PZP-proteinase complexes to the receptor by enzyme immunoassay. Our results clearly established that although both alpha-M-proteinase complexes specifically bind to LRP, PZP-chymotrypsin complexes bind to the receptor with lesser apparent affinity (Kd approximately equal 320 nM) than alpha2-M-chymotrypsin complexes (Kd approximately equal 40 nM). We also demonstrated that Ni2+ blocks the binding of alpha2-M-chymotrypsin complexes, but not PZP-chymotrypsin complexes, to LRP. These data suggest that the binding to LRP involves conformational differences between both alpha-Ms in a region immediately upstream of the carboxy terminal receptor recognition domain. The possibility that PZP-proteinase complexes interact with other receptors not available to alpha2-M-proteinase complexes could be considered.     

The carbohydrate-recognition domain of Dectin-2 is a C-type lectin with specificity for high mannose

We examined the carbohydrate-binding potential of the C-type lectin-like receptor Dectin-2 (Clecf4n). The carbohydrate-recognition domain (CRD) of Dectin-2 exhibited cation-dependent mannose/fucose-like lectin activity, with an IC(50) for mannose of approximately 20 mM compared to an IC(50) of 1.5 mM for the macrophage mannose receptor when assayed by similar methodology. The extracellular domain of Dectin-2 exhibited binding to live Candida albicans and the Saccharomyces-derived particle zymosan. This binding was completely abrogated by cation chelation and was competed by yeast mannans. We compared the lectin activity of Dectin-2 with that of two other C-type lectin receptors (mannose receptor and SIGNR1) known to bind fungal mannans. Both mannose receptor and SIGNR1 were able to bind bacterial capsular polysaccharides derived from Streptococcus pneumoniae, but interestingly they exhibited distinct binding profiles. The Dectin-2 CRD exhibited only weak interactions to some of these capsular polysaccharides, indicative of different structural or affinity requirements for binding, when compared with the other two lectins. Glycan array analysis of the carbohydrate recognition by Dectin-2 indicated specific recognition of high-mannose structures (Man(9)GlcNAc(2)). The differences in the specificity of these three mannose-specific lectins indicate that mannose recognition is mediated by distinct receptors, with unique specificity, that are expressed by discrete subpopulations of cells, and this further highlights the complex nature of carbohydrate recognition by immune cells.     

Recognition of IgG by Fcgamma receptor. The role of Fc glycosylation and the binding of peptide inhibitors

Recently determined crystal structures of the complex between immunoglobulin constant regions (Fc) and their Fc-respective receptors (FcR) have revealed the detailed molecular interactions of this receptor-ligand pair. Of particular interest is the contribution of a glycosylation at Asn(297) of the C(H)2 domain of IgG to receptor recognition. The carbohydrate moieties are found outside the receptor.Fc interface in all receptor.Fc complex structures. To understand the role of glycosylation in FcR recognition, the receptor affinities of a deglycosylated IgG1 and its Fc fragment were determined by solution binding studies using surface plasmon resonance. The removal of carbohydrates resulted in a non-detectable receptor binding to the Fc alone and a 15- to 20-fold reduction of the receptor binding to IgG1, suggesting that the carbohydrates are important in the function of the FcgammaRIII. Structurally, the carbohydrates attached to Asn(297) fill the cavity between the C(H)2 domains of Fc functioning equivalently as a hydrophobic core. This may stabilize a favorable lower hinge conformation for the receptor binding. The structure of the complex also revealed the dominance of the lower hinge region in receptor.Fc recognition. To evaluate the potential of designing small molecular ligands to inhibit the receptor function, four lower hinge peptides were investigated for their ability to bind to the receptor FcgammaRIII. These peptides bind specifically to FcgammaRIII with affinities 20- to 100-fold lower than IgG1 and are able to compete with Fc in receptor binding. The results of peptide binding illustrate new ways of designing therapeutic compounds to block Fc receptor activation.     

Bacterial adherence on replicas of sodium dodecyl sulfate-polyacrylamide gels

A method for determining which molecules in a complex mixture of proteins can function as bacterial receptors was devised. Salivary proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Bacteria that were metabolically labeled with 3H or externally labeled with 125I were incubated on the nitrocellulose replicas. After 18 h at 4 degrees C, the unbound cells were removed by repeated washing of the replicas, and the bands to which the radiolabeled bacteria bound were visualized by autoradiography. By this technique, Fusobacterium nucleatum, which adheres via carbohydrate residues on receptor molecules, and Staphylococcus aureus, which recognizes the peptide portion of fibronectin, were shown to bind specifically to their respective receptors. These results suggest that this method can be useful for profiling bacterial binding to either the carbohydrate or the protein portions of molecules present in complex mixtures, such as those composing biological fluids or tissue substrates. Structural specificities, such as recognition sequences formed by certain oligosaccharides, could be further investigated by adding the appropriate simple sugars, as well as oligosaccharide inhibitors, to the incubation medium. The latter approach is particularly important since most glycoproteins carry multiple N- and O-linked carbohydrate substituents that could serve as bacterial receptors.     

The Mammalian β-Adrenergic Receptor: Structural and Functional Characterization of the Carbohydrate Moiety

Abstract

Mammalian β-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of M_r ～64,000. Treatment of purified [¹²⁵I]-labeled hamster lung β-adrenergic receptor with α-mannosi-dase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [¹²⁵I]-labeled receptor with endo-glycosidase F results initially in the formation of a M_r ～57,000 peptide which is further converted to M_r ～49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [¹²⁵I]-labeled receptor reveals the presence of two complex type carbohydrate chains (～10% of which are fucosylated) on ～45% of the receptors. The remaining ～55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of β-adrenergic agonists and antagonists. Moreover, the ability of control, α-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, α-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleo-tide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the β-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.     

Stage-Specific Sampling by Pattern Recognition Receptors during Candida albicans Phagocytosis

Candida albicans is a medically important pathogen, and recognition by innate immune cells is critical for its clearance. Although a number of pattern recognition receptors have been shown to be involved in recognition and phagocytosis of this fungus, the relative role of these receptors has not been formally examined. In this paper, we have investigated the contribution of the mannose receptor, Dectin-1, and complement receptor 3; and we have demonstrated that Dectin-1 is the main non-opsonic receptor involved in fungal uptake. However, both Dectin-1 and complement receptor 3 were found to accumulate at the site of uptake, while mannose receptor accumulated on C. albicans phagosomes at later stages. These results suggest a potential role for MR in phagosome sampling; and, accordingly, MR deficiency led to a reduction in TNF-α and MCP-1 production in response to C. albicans uptake. Our data suggest that pattern recognition receptors sample the fungal phagosome in a sequential fashion.     

Mammalian beta-adrenergic receptors. Distinct glycoprotein populations containing high mannose or complex type carbohydrate chains

Mammalian beta-adrenergic receptor binding peptides can be visualized by covalently labeling them with the photoaffinity reagent p-azido-m-[125I]iodobenzylcarazolol followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The receptor peptides migrate as broad bands of Mr approximately equal to 62,000. In the present study, we examined the carbohydrate composition of the mammalian beta receptor through the use of specific exo- and endoglycosidases and lectin affinity chromatography. Treatment of p-azido-m-[125I]iodobenzylcarazolol-labeled beta2-adrenergic receptors from hamster lung or rat erythrocyte with the exoglycosidases neuraminidase and alpha-mannosidase provided evidence for the existence of both high mannose and complex type carbohydrate chains on beta 2-adrenergic receptors. The nonadditivity of the effect of sequential treatments with these enzymes suggested discrete populations of beta-adrenergic receptors containing either complex or high mannose type chains. Deglycosylation of receptor with endoglycosidase F results in a single labeled polypeptide at Mr = 49,000 for both systems. The same two populations of the beta receptors (high mannose or complex type chain) could also be fractionated by lectin affinity chromatography of solubilized p-azido-m-[125I]iodobenzylcarazolol-labeled receptors. The high mannose-containing receptors could be absorbed to and specifically eluted from concanavalin A-agarose. Those containing complex type carbohydrates could be adsorbed to and eluted from wheat germ agglutinin-agarose. Taken together, these data suggest that mammalian beta-adrenergic receptors contain both complex and high mannose type carbohydrate chains and that microheterogeneity of these chains likely explains the broad band pattern typically obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Macrophage Mannosyl Fucosyl Receptor: Its Role in Invasion of Virulent and Avirulent L. Donovani Promastigotes

The interaction of leishmania parasites with macrophages is known to be receptor mediated. Previous study from this laboratory (J. Parasitol. 82:632, 1996) showed the significant involvement of LPG and gp63 receptors in the recognition of virulent strains onto the macrophages. The role of carbohydrate receptors--the other major receptors besides LPG and gp63 receptors, in the recognition of both virulent (strains AG83 and GE1) and avirulent (strain UR6) leishmania onto the host macrophages has been the major focus of the present investigation. Various neoglycoproteins were used as efficient ligands to preblock the carbohydrate receptors on the macrophage surface. Similarly, various sugar specific lectins were used to preblock the corresponding carbohydrate ligands on the parasite surface. When these preblocked macrophages or parasites were used to study their mode of recognition, it was obvious from the findings that avirulent leishmania promastigotes possibly use the mannosyl fucosyl receptors (MFR) more avidly for their initial attachment and subsequent internalization into the macrophages whereas the virulent leishmania exhibits limited use of this receptor. When a macrophage-like cell line (J774), lacking in MFR, was purposely selected to test the previous findings, as expected, the attachment of avirulent promastigotes (UR6) onto the cell line was found to be negligible when compared to the peritoneal macrophages. Thus, it appears that avirulent leishmania promastigotes probably utilize MFR significantly for their initial recognition and subsequent internalization by macrophages.     

The sea urchin egg receptor for sperm: isolation and characterization of the intact, biologically active receptor

The species-specific binding of sea urchin sperm to the egg is mediated by an egg cell surface receptor. Although earlier studies have resulted in the cloning and sequencing of the receptor, structure/function studies require knowledge of the structure of the mature cell surface protein. In this study, we report the purification of this glycoprotein to homogeneity from a cell surface complex of Strongylocentrotus purpuratus eggs using lectin and ion exchange chromatography. Based on the yield of receptor it can be calculated that each egg contains approximately 1.25 x 10(6) receptor molecules on its surface. The receptor, which has an apparent M(r) of 350 kD, is a highly glycosylated transmembrane protein composed of approximately 70% carbohydrate. Because earlier studies on the partially purified receptor and on a pure, extracellular fragment of the receptor indicated that the carbohydrate chains were important in sperm binding, we undertook compositional analysis of the carbohydrate in the intact receptor. These analyses and lectin binding studies revealed that the oligosaccharide chains of the receptor are sulfated and that both N- and O-linked chains are present. Functional analyses revealed that the purified receptor retained biological activity; it inhibited fertilization in a species-specific and dose-dependent manner, and polystyrene beads coated with it bound to acrosome-reacted sperm in a species-specific manner. The availability of biochemical quantities of this novel cell recognition molecule opens new avenues to studying the interaction of complementary cell surface ligands in fertilization.     

Rat plasma murinoglobulin: isolation, characterization, and comparison with rat alpha-1- and alpha-2-macroglobulins

Murinoglobulin, a newly identified mouse plasma protein with trypsin-protein esterase activity (Saito, A. & Sinohara, H. (1985) J. Biol. Chem. 260, 775-781), was also found in rat plasma and purified to apparent homogeneity. The serum level of rat murinoglobulin was 14.1 mg/ml, amounting to 1/3 of the total serum globulin fraction. Rat murinoglobulin was a monomeric glycoprotein (Mr = 210,000) containing 12% carbohydrate. Rat plasma contained two isoforms of murinoglobulin, termed I and II, which showed complete immunological identity on double diffusion analysis using rabbit antiserum raised against isoform I or II. These antisera also showed partial cross-reactivity towards mouse murinoglobulin and rat alpha-1-macroglobulin but not towards rat or human alpha-2-macroglobulin. The chemical compositions, peptide mapping patterns and electrophoretic mobilities of the two isoforms resembled each other but clearly differed from those of rat alpha-1- or alpha-2-macroglobulin. Rat murinoglobulin inhibited the proteolytic activity of trypsin towards casein and remazol brilliant blue hide powder. The inhibition as to the latter substrate was greater than that as to the former. When molar ratios of inhibitor to trypsin were low, murinoglobulin and the two alpha-macroglobulins stimulated the amidolytic activity of trypsin towards a synthetic substrate. At higher ratios, however, murinoglobulin, but not the alpha-macroglobulins, inhibited the same activity. The trypsin-protein esterase activity of murinoglobulin and the two alpha-macroglobulins was impaired by a molar excess of soybean trypsin inhibitor. Murinoglobulin and the two alpha-macroglobulins were inactivated by methylamine with a concomitant unmasking of the thiol group. Murinoglobulin was much more sensitive to soybean trypsin inhibitor and methylamine than the two alpha-macroglobulins.     

Structural analysis and functional role of the carbohydrate component of somatostatin receptors.

SRIF receptors are membrane-bound glycoproteins. To structurally identify the carbohydrate components of SRIF receptors, solubilized rat brain SRIF receptors were subjected to lectin affinity chromatography. Solubilized SRIF receptors specifically bound to wheat germ agglutinin-lectin affinity columns but not to succinylated wheat germ agglutinin. This finding, as well as the ability of the solubilized receptor to interact with a Sambucus nigra L. lectin affinity column suggested that sialic acid residues are associated with SRIF receptors. The inability of the receptor to bind to concanavalin A, Dolichus biflorus agglutinin, Ulex europeaus I, and Jacalin lectin affinity columns suggests that high mannose, N-acetylgalactosamine, fucose, and O-linked carbohydrates are not associated with receptor. To investigate the functional role of the carbohydrate groups in brain SRIF receptors, specific sugars were selectively cleaved from SRIF receptors and the subsequent effect on the specific high affinity binding of the agonist [125I]MK 678 to SRIF receptors was determined. Treatment of the receptor with endoglycosidase D did not affect the specific binding of [125I] MK 678 to the solubilized SRIF receptors, consistent with the finding from lectin affinity chromatography that high mannose-type carbohydrate structures were not associated with SRIF receptors. Treatment of solubilized SRIF receptors with peptide-N-glycosidase F and endoglycosidases H and F reduced [125I]MK 678 binding to SRIF receptors indicating that either hybrid, or a combination of hybrid and complex N-linked carbohydrate structures, have a role in maintaining the receptor in a high affinity state for agonists. Treatment of solubilized SRIF receptors with neuraminidase from Vibrio cholera abolished high affinity agonist binding to the receptors, whereas treatment of the receptor with neuraminidase from Newcastle disease virus did not affect [125I]MK 678 binding to the receptor. These findings suggest that sialic acid residues in an alpha 2,6-configuration have a role in maintaining the SRIF receptor in a high affinity conformation for agonists. This is further indicated by studies on SRIF receptors in the pituitary tumor cell line, AtT-20. Treatment of AtT-20 cells in culture with neuraminidase (V. cholera) greatly reduces high affinity [125I] MK 678 binding sites, but did not alter the maximal ability of SRIF to inhibit forskolin-stimulated cAMP accumulation in intact AtT-20 cells. This finding suggests that the desialylated SRIF receptor is functionally active and remains coupled to GTP-binding proteins, but exhibits a reduced affinity for agonists. Treatment of AtT-20 cell membranes with neuraminidase from V. cholera was also able to greatly reduce the affinity of SRIF receptors for [125I]MK 678.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural analysis of the recognition mechanism of poly-N-acetyllactosamine by the human galectin-9 N-terminal carbohydrate recognition domain

Galectins are a family of beta-galactoside-specific lectins bearing a conserved carbohydrate recognition domain. Interactions between galectins and poly-N-acetyllactosamine sequences are critical in a variety of biological processes. Galectin-9, a member of the galectin family, has two carbohydrate recognition domains at both the N- and C-terminal regions. Here we report the crystal structure of the human galectin-9 N-terminal carbohydrate recognition domain in complex with N-acetyllactosamine dimers and trimers. These complex structures revealed that the galectin-9 N-terminal carbohydrate recognition domain can recognize internal N-acetyllactosamine units within poly-N-acetyllactosamine chains. Based on these complex structures, we propose two putative recognition modes for poly-N-acetyllactosamine binding by galectins.     

Identification of residues essential for carbohydrate recognition and cation dependence of the 46-kDa mannose 6-phosphate receptor

The 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) plays an essential role in the biogenesis of lysosomes by diverting newly synthesized mannose 6-phosphate (Man-6-P)-containing lysosomal enzymes from the secretory pathway to acidified endosomes. Previous crystallographic studies of the CD-MPR have identified 11 amino acids within its carbohydrate binding pocket. These residues were evaluated quantitatively by assaying the binding affinity of mutant receptors containing a single amino acid substitution toward a lysosomal enzyme. The results show that substitution of Gln-66, Arg-111, Glu-133, or Tyr-143 results in a >800-fold decrease in affinity, demonstrating these four amino acids are essential for carbohydrate recognition by the CD-MPR. Solution binding and surface plasmon resonance analyses demonstrated that the presence of Mn2+ enhanced the affinity of the CD-MPR for a lysosomal enzyme by 2- to 4-fold and increased the stoichiometry of the interaction between a heterogeneous population of a lysosomal enzyme and the receptor by approximately 3-fold. In contrast, substitution of Asp-103 results in a protein that no longer exhibits enhanced binding affinities or altered stoichiometry in the presence of cations, and electron spin resonance demonstrated that the D103S mutant exhibits a 6-fold lower affinity for Mn2+ than the wild-type receptor (Kd = 3.7 6 1.4 mM versus 0.6 6 0.1 mM). Chemical cross-linking revealed that Mn2+ influences the stoichiometry of interaction between the CD-MPR and lysosomal enzymes by increasing the oligomeric state of the receptor from dimer to higher order oligomers. Taken together, these studies provide the molecular basis for high affinity carbohydrate recognition by the CD-MPR. Furthermore, Asp-103 has been identified as the key residue which mediates the effects of divalent cations on the binding properties of the CD-MPR.     

Anti-self receptors

Rosetting between thymocytes and autologous erythrocytes is mediated by receptors on thymocytes that primarily recognize self H-2L molecules on erythrocytes. This paper describes preliminary attempts to chemically characterize the receptor and acceptor molecules involved in thisH-2-restricted interaction. On the basis of sugar inhibition studies and the sensitivity of the receptors and acceptors to protease and glycosidase treatments it appears that a protein receptor on thymocytes recognizes the carbohydrate portion of a glycoprotein on erythrocytes. Furthermore, the thymocyte receptor appears to recognize terminal D-galactose, D-mannose and sialic acid residues on a branched-chain carbohydrate structure on erythrocytes, with mouse strains of differentH-2 haplotype expressing carbohydrate structures that differ in the linkage of these three terminal sugars. These findings indicate thatH-2-restricted carbohydrate-protein interactions can occur between cells, a conclusion with important theoretical implications.     

Circular dichroic spectroscopy of non-human alpha-macroglobulins

Bovine, chicken and frog alpha-macroglobulins and ovomacroglobulin were studied by circular dichroic spectroscopy over the region 205-250 nm. All four spectra exhibited negative ellipticity with minima at about 215 nm similar to that reported for human alpha 2-macroglobulin. On reaction of the alpha-macroglobulins with trypsin, the spectrum of each of the four changed similarly. However, these proteins exhibited different conformational changes when treated with methylamine. These differences were exploited to determine which characteristics of alpha-macroglobulins correlate with changes in circular dichroic spectroscopy.     

T-cell recognition of glycolipids presented by CD1 proteins

The most well-known molecular paradigm of antigen recognition by T cells involves partial digestion of proteins to generate small peptides, which bind to major histocompatibility complex (MHC) proteins. Recent studies of CD1, an MHC class I homolog encoded outside the MHC, have revealed that it presents diverse glycolipids to T cells. The molecular mechanism for lipid antigen recognition involves insertion of the lipid portion of antigens into a hydrophobic groove to form CD1-lipid complexes, which contact T-cell receptors (TCRs). Here, we examine the known antigen structures presented by CD1, the majority of which have sugar moieties that are capable of interacting with TCRs. Recognition of carbohydrate epitopes is precise, and lipid-reactive T cells alter systemic immune responses in models of infectious and autoimmune disease. These findings provide a previously unrecognized mechanism by which the cellular immune system can recognize alterations in many types of carbohydrate structures.     

Clearance and binding of two electrophoretic "fast" forms of human alpha 2-macroglobulin

These studies explore the role of conformational change and exposed carbohydrate residues in the clearance of alpha 2-macroglobulin-trypsin (alpha 2M-T) complexes in the mouse. Human alpha 2-macroglobulin (alpha 2M) was purified and demonstrated to be homogeneous in the electrophoretic "slow" form. Two conformationally altered derivatives, alpha 2M-T and alpha 2-macroglobulin-methylamine (alpha 2M-MeNH2), were prepared and demonstrated to exist in the electrophoretic "fast" form. Radiolabeled alpha 2M-T and alpha 2M-MeNH2 were cleared rapidly with a half-life of 2-4 min following injection into mice. Radiolabeled native alpha 2M, however, remained in the circulation with a half-life of several hours. Both alpha 2M-T and alpha 2M-MeNH2 bound specifically to mouse peritoneal macrophages at 4 degrees C and occupancy of receptor sites increased with increasing time and radioligand concentration. Excess amounts of unlabeled alpha 2M-T or alpha 2M-MeNH2 cross-completed with trace amounts of the other in both clearance studies and binding assays, indicating that both derivatives were removed by the same receptor pathway. The clearance and binding of alpha 2M-T and alpha 2M-MeNH2 were not inhibited by excess amounts of unlabeled asialoorosomucoid, fucosyl-bovine serum albumin, mannosyl-BSA, or N-acetylglucosaminyl-BSA. Our results indicate that the clearance pathway removing alpha 2M-T complexes from the circulation recognizes a fundamental conformational change in alpha 2M secondary to protease binding, which can also be induced by exposure to methylamine. Therefore, other chemical or physical alterations that occur in alpha 2M upon binding trypsin, apart from the conformational change also present in alpha 2M-MeNH2, do not seem necessary for the recognition of alpha 2M-T by cells in the clearance pathway. In addition, this pathway appears distinct from several systems already described mediating clearance of glycoproteins through recognition of terminal galactose, fucose, N-acetylglucosamine, or mannose on oligosaccharide side chains.