Search PubMed⌕ Search

Biomedical subjects

P R Crocker

Publications and source records attributed to P R Crocker.

At least 19 recordsLinked to original sources
PubMed2000

Loss of N-glycolylneuraminic acid in human evolution. Implications for sialic acid recognition by siglecs.

The common sialic acids of mammalian cells are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans are an exception, because of a mutation in CMP-sialic acid hydroxylase, which occurred after our common ancestor with great apes. We asked if the resulting loss of Neu5Gc and increase in Neu5Ac in humans alters the biology of the siglecs, which are Ig superfamily members that recognize sialic acids. Human siglec-1 (sialoadhesin) strongly prefers Neu5Ac over Neu5Gc. Thus, humans have a higher density of siglec-1 ligands than great apes. Siglec-1-positive macrophages in humans are found primarily in the perifollicular zone, whereas in chimpanzees they also occur in the marginal zone and surrounding the periarteriolar lymphocyte sheaths. Although only a subset of chimpanzee macrophages express siglec-1, most human macrophages are positive. A known evolutionary difference is the strong preference of mouse siglec-2 (CD22) for Neu5Gc, contrasting with human siglec-2, which binds Neu5Ac equally well. To ask when the preference for Neu5Gc was adjusted in the human lineage, we cloned the first three extracellular domains of siglec-2 from all of the great apes and examined their preference. In fact, siglec-2 had evolved a higher degree of recognition flexibility before Neu5Gc was lost in humans. Human siglec-3 (CD33) and siglec-6 (obesity-binding protein 1) also recognize both Neu5Ac and Neu5Gc, and siglec-5 may have some preference for Neu5Gc. Others showed that siglec-4a (myelin-associated glycoprotein) prefers Neu5Ac over Neu5Gc. Thus, the human loss of Neu5Gc may alter biological processes involving siglec-1, and possibly, siglec-4a or -5.

Amino Acid Sequence↗
PubMed2000

Siglec-8. A novel eosinophil-specific member of the immunoglobulin superfamily.

We describe the characterization of siglec-8, a novel sialic acid-binding immunoglobulin-like lectin that is expressed specifically by eosinophils. A full-length cDNA encoding siglec-8 was isolated from a human eosinophil cDNA library. Siglec-8 is predicted to contain three extracellular immunoglobulin-like domains, a transmembrane region, and a cytoplasmic tail of 47 amino acids. The siglec-8 gene mapped on chromosome 19q13.33-41, closely linked to genes encoding CD33 (siglec-3), siglec-5, siglec-6, and siglec-7. When siglec-8 was expressed on COS cells or as a recombinant protein fused to the Fc region of human IgG(1), it was able to mediate sialic acid-dependent binding to human erythrocytes and to soluble sialoglycoconjugates. Using specific monoclonal antibodies, siglec-8 could be detected only on eosinophils and hence appears to be the first example of an eosinophil-specific transmembrane receptor.

Amino Acid Sequence↗
PubMed1999

Cell-specific glycoforms of sialoadhesin and CD45 are counter-receptors for the cysteine-rich domain of the mannose receptor.

We previously reported that CR-Fc, an Fc chimeric protein containing the cysteine-rich (CR) domain of the mannose receptor, binds to marginal zone metallophilic macrophages (Mo) and B cell areas in the spleen and to subcapsular sinus Mo in lymph nodes of naive mice (CR-Fc(+) cells). Several CR-Fc ligands were found in spleen and lymph node tissue lysates using ligand blots. In this paper we report the identification of two of these ligands as sialoadhesin (Sn), an Mo-specific membrane molecule, and the leukocyte common antigen, CD45. CR-Fc bound selectively to Sn purified from spleen and lymph nodes and to two low molecular weight isoforms of CD45 in a sugar-dependent manner. CR-Fc binding and non-binding forms of Sn, probably derived from CR-Fc(+) and CR-Fc(-) cells respectively, were selected from spleen lysates. Analysis of the glycan pool associated with the CR-Fc-binding form revealed the presence of charged structures resistant to sialidase, absent in the non-binding form, that could correspond to sulfated structures. These results confirm the identification of the CR region of the mannose receptor as a lectin. We also demonstrate that the same glycoprotein expressed in different cells of the same organ can display distinct sugar epitopes that determine its binding properties.

Animals↗
PubMed1999

Identification and characterization of a novel siglec, siglec-7, expressed by human natural killer cells and monocytes.

We describe the characterization of sialic acid-binding Ig-like lectin-7 (siglec-7), a novel member of the siglec subgroup of the immunoglobulin superfamily. A full-length cDNA encoding siglec-7 was isolated from a human primary dendritic cell cDNA library. Siglec-7 is predicted to contain three extracellular immunoglobulin-like domains that comprise an N-terminal V-set domain and two C2-set domains, a transmembrane region and a cytoplasmic tail containing two tyrosine residues embodied in immunoreceptor tyrosine-based inhibition motif-like motifs. Overall, siglec-7 exhibited a high degree of sequence similarity to genes encoding CD33 (siglec-3), siglec-5, OBBP1/siglec-6, and OBBP-like protein and mapped to the same region on chromosome 19q13.3. When siglec-7 was expressed on COS or Chinese hamster ovary cells, it was able to mediate high levels of sialic acid-dependent binding to human erythrocytes and soluble sialoglycoconjugates, suggesting that it may be involved in cell-cell interactions. Among human peripheral blood leukocytes, siglec-7 was found to be present at low levels on granulocytes, intermediate levels on monocytes, and relatively high levels on a major subset of natural killer cells and a minor subset of CD8(+) T cells. Immunoprecipitation experiments indicated that siglec-7 is expressed as a monomer of approximately 65 kDa.

3T3 Cells↗
PubMed1999

Molecular analysis of sialoside binding to sialoadhesin by NMR and site-directed mutagenesis.

The molecular interactions between sialoadhesin and sialylated ligands have been investigated by using proton NMR. Addition of ligands to the 12 kDa N-terminal immunoglobulin-like domain of sialoadhesin result in resonance shifts in the protein spectrum that have been used to determine the affinities of sialoadhesin for several sialosides. The results indicate that alpha2, 3-sialyl-lactose and alpha2,6-sialyl-lactose bind respectively 2- and 1.5-fold more strongly than does alpha-methyl-N-acetylneuraminic acid (alpha-Me-NeuAc). The resonances corresponding to the methyl protons within the N-acetyl moiety of sialic acid undergo upfield shifting and broadening during titrations, reflecting an interaction of this group with Trp2 in sialoadhesin as observed in co-crystals of the terminal domain with bound ligand. This resonance shift was used to measure the affinities of mutant and wild-type forms of sialoadhesin in which the first three domains are fused to the Fc region of human IgG1. Substitution of Arg97 by alanine completely abrogated measurable interaction with alpha-Me-NeuAc, whereas a conservative substitution with lysine resulted in a 10-fold decrease in affinity. These results provide the first direct measurement of the affinity of sialoadhesin for sialosides and confirm the critical importance of the conserved arginine in interactions between sialosides and members of the siglec family of sialic acid-binding, immunoglobulin-like lectins.

Binding Sites↗
PubMed1999

Sialoadhesin-positive host macrophages play an essential role in graft-versus-leukemia reactivity in mice.

We recently established an effective immune T-cell-mediated graft-versus-leukemia (GVL) murine model system in which complete tumor remissions were achievable even in advanced metastasized cancer. We now describe that this T-cell-mediated therapy is dependent on host macrophages expressing the lymphocyte adhesion molecule sialoadhesin (Sn). Depletion of Kupffer cells in tumor-bearing mice during adoptive immunotherapy (ADI) or the treatment of these animals with anti-Sn monoclonal antibodies led to complete or partial inhibition of the immune T-cell-mediated therapeutic effect. Furthermore, Sn+ host macrophages in livers formed clusters during ADI with donor CD8 T cells. To test for a possible antigen presentation function of these macrophages, we used as an in vitro model the antigen beta-galactosidase for which a dominant major histocompatibility complex (MHC) class I Ld-restricted peptide epitope is known to be recognized by specific CD8 cytotoxic T lymphocytes (CTL). We demonstrate that purified Sn+ macrophages can process exogenous beta-galactosidase and stimulate MHC class I peptide-restricted CTL responses. Thus, Sn+ macrophages, which are significantly increased in the liver after ADI, may process tumor-derived proteins via the MHC class I pathway as well as via the MHC class II pathway, as shown previously, and present respective peptide epitopes to CD8 as well as to CD4 immune T cells, respectively. The synergistic interactions observed before between immune CD4 and CD8 T cells during ADI could thus occur in the observed clusters with Sn+ host macrophages.

Animals↗
PubMed1999

Identification of CD22 ligands on bone marrow sinusoidal endothelium implicated in CD22-dependent homing of recirculating B cells.

CD22 is a B cell-specific transmembrane protein known to function as a negative regulator of B cell signaling. It has also been implicated in cell adhesion through recognition of alpha2,6-linked sialic acids on glycans of target cells. Previous studies showed that CD22-deficient mice had a strongly reduced population of mature recirculating B cells in the bone marrow despite normal B cell development. Using a soluble recombinant form of the receptor (CD22-Fc), we demonstrate here that sialylated ligands for CD22 are expressed on sinusoidal endothelial cells of murine bone marrow but not on endothelial cells in other tissues examined. Injection of CD22-Fc revealed that the CD22 ligands in the bone marrow were accessible to the circulation. Treatment of mice with either CD22-Fc or affinity-purified anti-CD22 antibody led to an approximately 50% reduction in mature recirculating B cells in the bone marrow without affecting numbers in the spleen. Finally, consistent with the notion that CD22 is a homing receptor, we show that compared with wild-type mice, CD22-deficient animals have a lower number of immunoglobulin M-secreting plasma cells in the bone marrow.

Animals↗
PubMed1999

Sialic acid binding receptors (siglecs) expressed by macrophages.

Sialic acids are structurally and topographically well-suited to function as ligands in cellular recognition events. Sialoadhesin (Sn) is a sialic acid binding receptor uniquely expressed by macrophage subsets. It is a member of the immunoglobulin (Ig) superfamily with 17 extracellular domains. Sn is a prototypical member of the siglec family of sialic acid binding proteins, which includes CD22, myelin-associated glycoprotein, CD33, and siglec-5. These membrane proteins are involved in discrete functions in the hemopoietic, immune, and nervous systems. The sialic acid binding region of siglecs is localized within the membrane-distal, amino-terminal domain and in the case of Sn, it has been characterized in atomic detail by X-ray crystallography, nuclear magnetic resonance, and site-directed mutagenesis. Our studies on Sn indicate that this receptor is likely to function as a macrophage accessory molecule in a variety of cell-cell and cell-extracellular matrix interactions. CD33 and siglec-5 are also expressed on macrophage subsets as well as other myeloid cells. However, unlike Sn, the properties of these molecules indicate a predominant role in signaling functions rather than in cell-cell interactions.

Amino Acid Sequence↗
PubMed1999

PECAM-1 and leukosialin (CD43) expression correlate with heightened inflammation in rat adjuvant-induced arthritis.

A hallmark of both adjuvant-induced arthritis (AIA) and rheumatoid arthritis is chronic joint inflammation characterized by ingress of leukocytes into the inflamed synovial tissue. The timing of expression of adhesion molecules, which govern the ingress of leukocytes, is important in the orchestration of an inflammatory response. We examined the expression of vascular cell adhesion molecule-1 (VCAM-1), sialo adhesin, platelet and endothelial cell adhesion molecule-1 (PECAM-1), and leukosialin (CD43) in AIA, starting at adjuvant injection (day 0), through the peak of inflammation (day 18 postadjuvant injection), until day 54. VCAM-1 is constitutively expressed on the lining layer and ECs and its expression levels do not change throughout the progression of AIA. Sialoadhesin synovial tissue lining cell expression is decreased after adjuvant injection. In contrast, PECAM-1 expression is increased on synovial tissue lining cells on day 7 and is elevated through day 54 (peaking on day 54 with six-fold more cells expressing PECAM-1). PECAM-1 expression on endothelial cells peaks on day 7 with three-fold more cells expressing it, while on macrophages expression maximizes on day 25 with six-fold more cells expressing PECAM-1. CD43 expression is increased on synovial tissue lining cells, macrophages, neutrophils, and lymphocytes on days 18 and 25, before going back to basal levels. The increased expression of PECAM-1 and CD43 on leukocytes at the height of inflammation in AIA suggests important roles for these adhesion molecules in potentially binding their EC ligands resulting in leukocyte ingress into the synovial tissue.

Animals↗
PubMed1999

Macrophage-tumour cell interactions: identification of MUC1 on breast cancer cells as a potential counter-receptor for the macrophage-restricted receptor, sialoadhesin.

In many carcinomas, infiltrating macrophages are commonly found closely associated with tumour cells but little is known concerning the nature or significance of adhesion molecules involved in these cellular interactions. Here we demonstrate in primary human breast cancers that sialoadhesin (Sn), a macrophage-restricted adhesion molecule, is frequently expressed on infiltrating cells that often make close contact with breast carcinoma cells. To determine whether Sn could act as a specific receptor for ligands on breast cancer cell lines, binding assays were performed with a recombinant form of the protein fused to the Fc portion of human immunoglobulin G1 (IgG1) (Sn-Fc). Sn-Fc was found to bind specifically and in a sialic acid-dependent manner to the breast cancer cell lines MCF-7, T47.D and BT-20 both in solid- and solution-phase binding assays. To investigate the nature of the sialoglycoproteins recognized by Sn on breast cancer cells, MCF-7 cells were labelled with [6-3H]glucosamine. Following precipitation with Sn-Fc, a major band of approximately 240000 MW was revealed, which was shown in reprecipitation and Western blotting experiments to be the epithelial mucin, MUC1.

Blotting, Western↗
PubMed1999

A six-year longitudinal study of the relationship of physical activity to bone mineral accrual in growing children: the university of Saskatchewan bone mineral accrual study.

To investigate the influence of physical activity on bone mineral accrual during the adolescent years, we analyzed 6 years of data from 53 girls and 60 boys. Physical activity, dietary intakes, and anthropometry were measured every 6 months and dual-energy X-ray absorptiometry scans of the total body (TB), lumbar spine (LS), and proximal femur (Hologic 2000, array mode) were collected annually. Distance and velocity curves for height and bone mineral content (BMC) were fitted for each child at several skeletal sites using a cubic spline procedure, from which ages at peak height velocity (PHV) and peak BMC velocity (PBMCV) were identified. A mean age- and gender-specific standardized activity (Z) score was calculated for each subject based on multiple yearly activity assessments collected up until age of PHV. This score was used to identify active (top quartile), average (middle 2 quartiles), or inactive (bottom quartile) groups. Two-way analysis of covariance, with height and weight at PHV controlled for, demonstrated significant physical activity and gender main effects (but no interaction) for PBMCV, for BMC accrued for 2 years around peak velocity, and for BMC at 1 year post-PBMCV for the TB and femoral neck and for physical activity but not gender at the LS (all p < 0.05). Controlling for maturational and size differences between groups, we noted a 9% and 17% greater TB BMC for active boys and girls, respectively, over their inactive peers 1 year after the age of PBMCV. We also estimated that, on average, 26% of adult TB bone mineral was accrued during the 2 years around PBMCV.

Absorptiometry, Photon↗
PubMed1998

Characterization of siglec-5, a novel glycoprotein expressed on myeloid cells related to CD33.

We describe the characterization of siglec-5 (sialic acid-binding Ig-like lectin-5), a novel transmembrane member of the immunoglobulin superfamily, highly related to the myeloid antigen, CD33. A full-length cDNA encoding siglec-5 was isolated from a human activated monocyte cDNA library. Sequencing predicted that siglec-5 contains four extracellular immunoglobulin-like domains, the N-terminal two of which are 57% identical to the corresponding region of CD33. The cytoplasmic tail is also related to that of CD33, containing two tyrosine residues embodied in immunoreceptor tyrosine-based inhibitory motif-like motifs. The siglec-5 gene was shown to map to chromosome 19q13.41-43, closely linked to the CD33 gene. When siglec-5 was expressed on COS cells or as a recombinant protein fused to the Fc region of human IgG1, it was able to mediate sialic acid-dependent binding to human erythrocytes and soluble glycoconjugates, suggesting that it may be involved in cell-cell interactions. By using specific antibodies, siglec-5 was found to have an expression pattern distinct from that of CD33, being present at relatively high levels on neutrophils but absent from leukemic cell lines representing early stages of myelomonocytic differentiation. Western blot analysis of neutrophil lysates indicated that siglec-5 exists as a disulfide-linked dimer of approximately 140 kD.

Amino Acid Sequence↗
PubMed1998

Myelin-associated glycoprotein binding to gangliosides. Structural specificity and functional implications.

Myelin-associated glycoprotein (MAG), which mediates certain myelin-neuron cell-cell interactions, is a lectin that binds to sialylated glycoconjugates. Gangliosides, the most abundant sialylated glycoconjugates in the brain, may be the functional neuronal ligands for MAG. Cells engineered to express MAG on their surface adhered specifically to gangliosides bearing an alpha 2,3-linked N-acetylneuraminic acid on a terminal galactose, with the following relative potency: GQ1b alpha >> GD1a, GT1b >> GM3, GM4 (GM1, GD1b, GD3, and GQ1b did not support adhesion). MAG binding was abrogated by modification of the carboxylic acid, any hydroxyl, or the N-acetyl group of the ganglioside's N-acetylneuraminic acid moiety. Related immunoglobulin (Ig) superfamily members either failed to bind gangliosides (CD22) or bound with less stringent specificity (sialoadhesin), whereas a modified form of MAG (bearing three of its five extra-cellular Ig-like domains) bound only GQ1b alpha. Enzymatic removal of sialic acids from the surface of intact nerve cells altered their functional interaction with myelin. These data are consistent with a role for gangliosides in MAG-neuron interactions.

Animals↗
PubMed1998

Crystal structure of the N-terminal domain of sialoadhesin in complex with 3' sialyllactose at 1.85 A resolution.

The structure of the functional N-terminal domain from the extracellular region of the cell surface receptor sialoadhesin has been determined in complex with the oligosaccharide 3' sialyllactose. This provides structural information for the siglec family of proteins. The structure conforms to the V-set immunoglobulin-like fold but contains several distinctive features, including an intra-beta sheet disulphide and a splitting of the standard beta strand G into two shorter strands. These novel features appear important in adapting the V-set fold for sialic acid-mediated recognition. Analysis of the complex with 3'sialyllactose highlights three residues, conserved throughout the siglec family, as key features of the sialic acid-binding template. The complex is representative of the functional recognition interaction with carbohydrate and as such provides detailed information for a heterotypic cell adhesion interaction.

Animals↗
PubMed1998

Maintenance of granulocyte numbers during acute peritonitis is defective in galectin-3-null mutant mice.

Galectin-3, also known as the macrophage marker Mac-2, is a member of a family of structurally related animal lectins that exhibit specificity for beta-galactosides. In order to investigate the role of galectin-3 in acute inflammation, we have compared the number of leucocytes present in the peritoneal cavity of wild type and galectin-3 null mutant mice after intraperitoneal (i.p.) injection of thioglycolate broth. At day 1 after injection, we found no difference in the recruitment of mononuclear phagocytes and granulocytes to the peritoneal cavity. However, 4 days after thioglycolate injection, galectin-3 mutant mice exhibited a significantly reduced number of recoverable granulocytes compared to wild-type animals. As mutant granulocytes did not exhibit an accelerated rate of apoptosis and their uptake by macrophages appeared to be unaffected by the mutation, the phenotype described here suggests that galectin-3 participates in an additional level of control during the resolution of acute inflammation.

Acute Disease↗
PubMed1998

A streptavidin-based neoglycoprotein carrying more than 140 GT1b oligosaccharides: quantitative estimation of the binding specificity of murine sialoadhesin expressed on CHO cells.

We prepared a streptavidin-based neoglycoprotein which carries more than 140 GT1b oligosaccharides. GT1b oligosaccharides were covalently coupled to streptavidin by reductive amination, yielding a monomer form of streptavidin carrying 13 oligosaccharides. The monomer form of glycosylated streptavidin was polymerized with biotinylated-bovine serum albumin, which yielded a polymer carrying more than 140 oligosaccharides. Both the monomer and the polymer bound to Chinese hamster ovary cells expressing murine sialoadhesin. The relative binding potencies determined with the polymer, monomer, and free GT1b oligosaccharides were 3,500, 83, and 1, respectively, indicating that an increase in the number of oligosaccharide ligands is critical for high avidity. The high avidity of the polymer enabled us to develop a sensitive and quantitative binding assay, and the assay was applied to characterization of the binding specificity of sialoadhesin. The polymer binding was inhibited by various gangliosides, the order of the inhibitory potencies being GM3 (IC50 = 40 microM) > GD1a (100 microM) > sialylparagloboside (120 microM) > GT1b (310 microM) > GM2 (640 microM) > GM4 (2,100 microM) > GD1b>LacCer = GM1 = paragloboside (no inhibition). These results indicate that the binding specificity is comparable to that reported, i.e. the determinant structure is NeuAcalpha2-3Galbeta1-linked to either 3GalNAc, 3(4)GlcNAc, or 4Glc, and that the oligosaccharide structure on the polymer is properly presented to sialoadhesin on the cell surface. To determine the precise requirement of the NeuAc structure for binding, NeuAc of GM3 was converted into various derivatives, the inhibitory potencies of which were examined; i.e. GM3 containing NeuAc, IC50 = 40 microM; C7- or C8-aldehyde, 500 microM; C7- or C8-alcohol, 700 microM; C1-alcohol, 2,000 microM; C1-amide, 2,200 microM; and NeuGc,>3,000 microM. These results confirmed the requirement of the hydroxyl group at C9 and/or C8, the carboxyl group at C1, and the methyl group of the N-acetyl residue of NeuAc in a quantitative manner. Thus, this streptavidin-based neoglycoprotein is a useful multivalent glycoprobe, which exhibits high affinity and specificity to murine sialoadhesin on the cell surface.

Animals↗
PubMed1997

Myelin-associated glycoprotein interacts with neurons via a sialic acid binding site at ARG118 and a distinct neurite inhibition site.

Inhibitory components in myelin are largely responsible for the lack of regeneration in the mammalian CNS. Myelin-associated glycoprotein (MAG), a sialic acid binding protein and a component of myelin, is a potent inhibitor of neurite outgrowth from a variety of neurons both in vitro and in vivo. Here, we show that MAG's sialic acid binding site is distinct from its neurite inhibitory activity. Alone, sialic acid-dependent binding of MAG to neurons is insufficient to effect inhibition of axonal growth. Thus, while soluble MAG-Fc (MAG extracellular domain fused to Fc), a truncated form of MAG-Fc missing Ig-domains 4 and 5, MAG(d1-3)-Fc, and another sialic acid binding protein, sialoadhesin, each bind to neurons in a sialic acid- dependent manner, only full-length MAG-Fc inhibits neurite outgrowth. These results suggest that a second site must exist on MAG which elicits this response. Consistent with this model, mutation of arginine 118 (R118) in MAG to either alanine or aspartate abolishes its sialic acid-dependent binding. However, when expressed at the surface of either CHO or Schwann cells, R118-mutated MAG retains the ability to inhibit axonal outgrowth. Hence, MAG has two recognition sites for neurons, the sialic acid binding site at R118 and a distinct inhibition site which is absent from the first three Ig domains.

Amino Acid Sequence↗