Glycosylation-dependent Cell Adhesion Molecule Research Articles

Parous mammary glands exhibit distinct alterations in gene expression and proliferation responsiveness to carcinogenic stimuli in Lewis rats

Early full-term pregnancy affords lifetime protection against the development of breast cancer. Parity-induced protection can be reproduced in a carcinogen-induced rat mammary carcinoma model, but the molecular mechanisms of this protection against carcinogenic stimuli in rat mammary glands have not been fully characterized. To gain a better understanding of these molecular mechanisms, we used an oligonucleotide microarray to examine gene expression in parous and age-matched virgin (AMV) mammary glands of Lewis rats before and after carcinogen (N-methyl-N-nitrosourea; MNU) treatment. Parous mammary glands before MNU treatment showed up-regulation of multiple differentiation-related genes, such as whey acidic protein (Wap), casein beta (Csn2), casein gamma (Csng), lipopolysaccharide binding protein (Lbp), secreted phosphoprotein 1 (Spp1) and glycosylation-dependent cell adhesion molecule 1 (Glycam1). Also, parous mammary glands before MNU treatment exhibited down-regulation of growth-related genes such as regenerating islet-derived 3 alpha (Reg3a), mesothelin (Msln), insulin-like growth factor 2 (Igf2) and insulin-like growth factor binding protein 4 (Igfbp4). After MNU treatment, AMV mammary glands exhibited up-regulation of growth-related genes, such as Msln, cell division cycle 2 homolog A (Cdc2a), Igf2, Igfbp4, stathmin 1 (Stmn1) and homeobox, msh-like 1 (Msx1), whereas expression of these genes remained low in parous mammary glands. AMV mammary glands also exhibited marked up-regulation of Cdc2a and Stmn1 in response to MNU. After MNU treatment, the PCNA labeling index increased significantly in AMV mammary epithelial cells (13.7+/-1.1%), but remained low in parous mammary glands (3.6+/-0.4%). The response of AMV mammary glands to carcinogenic stimuli includes up-regulation of growth-related genes and increased cell proliferation. The lack of a similar response in parous mammary glands may explain parity-induced protection against mammary tumor development.

IκB Kinase Complex α Kinase Activity Controls Chemokine and High Endothelial Venule Gene Expression in Lymph Nodes and Nasal-Associated Lymphoid Tissue

The lymphotoxin (LT) beta receptor plays a critical role in secondary lymphoid organogenesis and the classical and alternative NF-kappaB pathways have been implicated in this process. IKKalpha is a key molecule for the activation of the alternative NF-kappaB pathway. However, its precise role and target genes in secondary lymphoid organogenesis remain unknown, particularly with regard to high endothelial venules (HEV). In this study, we show that IKKalpha(AA) mutant mice, who lack inducible kinase activity, have hypocellular lymph nodes (LN) and nasal-associated lymphoid (NALT) tissue characterized by marked defects in microarchitecture and HEV. In addition, IKKalpha(AA) LNs showed reduced lymphoid chemokine CCL19, CCL21, and CXCL13 expression. IKKalpha(AA) LN- and NALT-HEV were abnormal in appearance with reduced expression of peripheral node addressin (PNAd) explained by a severe reduction in the HEV-associated proteins, glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), and high endothelial cell sulfotransferase, a PNAd-generating enzyme that is a target of LTalphabeta. In this study, analysis of LTbeta(-/-) mice identifies GlyCAM-1 as another LTbeta-dependent gene. In contrast, TNFRI(-/-) mice, which lose classical NF-kappaB pathway activity but retain alternative NF-kappaB pathway activity, showed relatively normal GlyCAM-1 and HEC-6ST expression in LN-HEV. In addition, in this communication, it is demonstrated that LTbetaR is prominently expressed on LN- and NALT-HEV. Thus, these data reveal a critical role for IKKalpha in LN and NALT development, identify GlyCAM-1 and high endothelial cell sulfotransferase as new IKKalpha-dependent target genes, and suggest that LTbetaR signaling on HEV can regulate HEV-specific gene expression.

A stroma-derived defect in NF-kappaB2-/- mice causes impaired lymph node development and lymphocyte recruitment.

The NF-kappaB family of transcription factors is vital to all aspects of immune function and regulation in both the hemopoietic and stromal compartments of immune environments. Recent studies of mouse models deficient for specific members of the NF-kappaB family have revealed critical roles for these proteins in the process of secondary lymphoid tissue organogenesis. In this study, we investigate the role of NF-kappaB family member NF-kappaB2 in lymph node development and lymphocyte recruitment. Inguinal lymph nodes in nfkappab2(-/-) mice are reduced in size and cellularity, most notably in the B cell compartment. Using in vitro and in vivo lymph node grafting assays, we show that the defect resides in the stromal compartment. Further examination of the nfkappab2(-/-) inguinal lymph nodes revealed that expression of peripheral node addressin components CD34 and glycosylation-dependent cell adhesion molecule-1 along with the high endothelial venule-restricted sulfotransferase HEC-GlcNAc6ST was markedly reduced. Furthermore, expression of the lymphocyte homing chemokines CCL19, CCL21, and CXCL13 was down-regulated. These data highlight the role of NF-kappaB2 in inguinal lymph node organogenesis and recruitment of lymphocytes to these organs due to its role in up-regulation of essential cell adhesion molecules and chemokines, while suggesting a potential role for NF-kappaB2 in organization of lymph node endothelium.

Structure and expression of goat GLYCAM1 gene: lactogenic-dependent expression in ruminant mammary gland and interspecies conservation of the proximal promoter

A macroarray approach used to list genes differentially expressed in goat mammary gland (gestation vs. lactation), other than milk protein genes, allowed us to detect the Glycosylation-dependent Cell Adhesion Molecule 1 ( GLYCAM1) gene. GLYCAM1, a member of the glycoprotein mucin family, is a component of the milk fat globule membrane (MFGM). Its complete cDNA and gene sequences were determined and it was mapped by fluorescent in situ hybridization (FISH) on goat and cattle chromosome 5 (CHI5q21 and BTA5q21), and on sheep chromosome 3 (OAR3q21). Northern blot analyses confirmed its differential expression during the development and differentiation of the mammary gland of ruminants with a significantly higher mRNA amount during lactation than during pregnancy. An experimental in vivo induction model for lactation, developed by Kann et al. [J. Endocrinol. 160 (1999) 265], showed that the expression of GLYCAM1 is hormonally regulated in the mammary gland of ewes. Interspecies comparison of the gene promoter revealed the evolutionary conservation of a short proximal nucleotide sequence encompassing several transcription factor binding sites that could mediate the above-mentioned hormonal regulation.

Two tandemly linked interferon-gamma-activated sequence elements in the promoter of glycosylation-dependent cell adhesion molecule 1 gene synergistically respond to prolactin in mouse mammary epithelial cells.

Previously, we reported that glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1) was a novel target for prolactin (PRL) in the mouse mammary gland. However, the signaling pathway by which PRL regulates GlyCAM 1 expression has not been specified. In the present study, we showed that PRL induced GlyCAM 1 expression in primary mammary epithelial cells of mice through the Janus kinase 2/signal transducer and activator of transcription 5 (Stat5) pathway. Deletion and site-directed mutagenesis analyses of the GlyCAM 1 promoter demonstrated that the two tandemly linked Stat5 binding sites [interferon-gamma-activated sequence 1 and -2 (GAS1 and GAS2)] in the proximal promoter region were crucial and synergistically responded to PRL. GAS2, a consensus GAS site, was essential and, by itself, weakly responded to PRL, whereas GAS1, a nonconsensus site, failed to respond to PRL but was indispensable for the maximal activity of the GlyCAM 1 promoter. Gel shift assays showed that probe containing GAS1 and GAS2 bound two Stat5 complexes, which represent Stat5 dimer and tetramer, respectively, while GAS2, by itself, bound Stat5 as a dimer only, and GAS1 showed no apparent binding activity. Interruption of tetramer formation by mutation of a tryptophan to alanine (W37A), and a leucine to serine (L83S) in the N terminus of Stat5A attenuated the synergistic effect between the two tandemly linked GAS sites. Overexpression of W37A and L83S mutants in primary mammary epithelial cells suppressed endogenous GlyCAM 1 expression.

Expression of potential lymphocyte trafficking mediator molecules in the mammary gland.

The mammary gland performs a variety of immunological functions, including protecting itself from mastitis and protecting neonates from infectious agents. Several molecules that mediate lymphocyte trafficking in the immune system are also expressed in the mammary gland. This review is focused on the immunological function of these molecules, especially glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the mammary gland. GlyCAM-1 is expressed in the lactating mouse mammary gland. Endothelial cells produce this protein and secrete it into milk. The glycosylated modification of mammary gland GlyCAM-1 is different from that of the lymph nodes, and lacks the binding ability for L-selectin on lymphocytes. GlyCAM-1 in the mammary gland is not involved in lymphocyte migration, and probably has another function besides that of the lymph nodes. MAdCAM-1 is expressed on endothelial cells of small venules around mouse mammary lobules during lactation. This molecule has the ability to interact with alpha4beta7 integrin on lymphocytes and mediates lymphocyte recruitment to the mammary gland. The density of beta7+/CD3+ T-cells is correlated with the density of the MAdCAM-1-stained area, suggesting that MAdCAM-1 may mediate the migration of these cells. In contrast, there is no relationship between MAdCAM-1 expression and the number of beta7+/c-IgA+ B-cells, implying that some other factor is involved in lymphocyte migration to the mammary gland. Chemokines, such as IL-8, GRO-alpha, MCP-1, RANTES and MEC, have been detected in human and mouse mammary glands. Although little information is available, these molecules may contribute to lymphocyte migration to the mammary gland.

Assessment of glycosylation-dependent cell adhesion molecule 1 as a correlate of allergen-stimulated lymph node activation

Early changes in gene expression have been identified by cDNA microarray technology. Analysis of draining auricular lymph node tissue sampled at 48 h following exposure to the potent contact allergen 2,4-dinitrofluorobenzene (DNFB) provided examples of up- and down-regulated genes, including onzin and guanylate binding protein 2, and glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), respectively. Allergen-induced changes in these three genes were confirmed in dose–response and kinetic analyses using Northern blotting and/or reverse transcription-polymerase chain reaction techniques. The results confirmed that these genes are robust and relatively sensitive markers of early changes provoked in the lymph node by contact allergen. Upon further investigation, it was found that altered expression of the adhesion molecule GlyCAM-1 was not restricted to treatment with DNFB. Topical sensitization of mice to a chemically unrelated contact allergen, oxazolone, was also associated with a decrease in the expression of mRNA for GlyCAM-1. Supplementary experiments revealed that changes in expression of this gene are independent of the stimulation by chemical allergens of proliferative responses by draining lymph node cells. Taken together these data indicate that the expression of GlyCAM-1 is down-regulated rapidly following epicutaneous treatment of mice with chemical allergens, but that this reduction is associated primarily with changes in lymph node cell number, or some other aspect of lymph node activation, rather than proliferation.

Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongation

Endometrial glands are necessary for conceptus implantation and growth. In the ovine uterine gland knockout (UGKO) model, blastocysts hatch normally but fail to survive or elongate. This peri-implantation defect in UGKO ewes may be due to the absence of endometrial glands or, alternatively, to the lack of certain epithelial adhesion molecules or the inability of the endometrium to respond to signals from the conceptus. Two studies were performed to examine these hypotheses. In study one, normal (n = 8) and UGKO (n = 12) ewes were mated at oestrus (day 0) with intact rams and their uteri were flushed 14 days after oestrus. Normal ewes (n = 4) were also flushed on 14 days after oestrus. Uterine flushes from bred normal ewes contained filamentous conceptuses (n = 7 of 8), whereas those from UGKO ewes contained no conceptus (n = 5 of 12), a growth-retarded, tubular conceptus (n = 6 of 12), or a fragmented, filamentous conceptus (n = 1 of 12). In all groups, expression of mucin 1 and integrin alpha(v), alpha(5), beta(3) and beta(5) was localized at the apical surface of the endometrial luminal epithelium with no detectable differences between normal and UGKO ewes. Uterine flushes from pregnant ewes, but not cyclic or UGKO ewes, contained abundant immunoreactive interferon tau and the cell adhesion proteins, osteopontin and glycosylation-dependent cell adhesion molecule one. In study two, UGKO ewes were fitted with uterine catheters 5 days after oestrus, infused with recombinant ovine interferon tau or control proteins from 11 to 15 days after oestrus, and underwent hysterectomy 16 days after oestrus. Expression of several interferon tau-stimulated genes (ISG17, STAT1, STAT2 and IRF-1) was increased in the endometrium from interferon tau-infused UGKO ewes. These results support the hypothesis that the defects in conceptus elongation and survival in UGKO ewes are due to the absence of endometrial glands and their secretions rather than to alterations in expression of anti-adhesive or adhesive molecules on the endometrial luminal epithelium or to the responsiveness of the endometrium to the conceptus pregnancy recognition signal.

Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongation

Endometrial glands are necessary for conceptus implantation and growth. In the ovine uterine gland knockout (UGKO) model, blastocysts hatch normally but fail to survive or elongate. This peri-implantation defect in UGKO ewes may be due to the absence of endometrial glands or, alternatively, to the lack of certain epithelial adhesion molecules or the inability of the endometrium to respond to signals from the conceptus. Two studies were performed to examine these hypotheses. In study one, normal (n = 8) and UGKO (n = 12) ewes were mated at oestrus (day 0) with intact rams and their uteri were flushed 14 days after oestrus. Normal ewes (n = 4) were also flushed on 14 days after oestrus. Uterine flushes from bred normal ewes contained filamentous conceptuses (n = 7 of 8), whereas those from UGKO ewes contained no conceptus (n = 5 of 12), a growth-retarded, tubular conceptus (n = 6 of 12), or a fragmented, filamentous conceptus (n = 1 of 12). In all groups, expression of mucin 1 and integrin alpha(v), alpha(5), beta(3) and beta(5) was localized at the apical surface of the endometrial luminal epithelium with no detectable differences between normal and UGKO ewes. Uterine flushes from pregnant ewes, but not cyclic or UGKO ewes, contained abundant immunoreactive interferon tau and the cell adhesion proteins, osteopontin and glycosylation-dependent cell adhesion molecule one. In study two, UGKO ewes were fitted with uterine catheters 5 days after oestrus, infused with recombinant ovine interferon tau or control proteins from 11 to 15 days after oestrus, and underwent hysterectomy 16 days after oestrus. Expression of several interferon tau-stimulated genes (ISG17, STAT1, STAT2 and IRF-1) was increased in the endometrium from interferon tau-infused UGKO ewes. These results support the hypothesis that the defects in conceptus elongation and survival in UGKO ewes are due to the absence of endometrial glands and their secretions rather than to alterations in expression of anti-adhesive or adhesive molecules on the endometrial luminal epithelium or to the responsiveness of the endometrium to the conceptus pregnancy recognition signal.

Linkage mapping of microsatellites associated with the glycosylation dependent cell adhesion molecule (GLYCAM1) and thyroid hormone receptor alpha chain (THRA) genes to ovine chromosomes 3 and 11.

Linkage mapping of microsatellites associated with the glycosylation dependent cell adhesion molecule (GLYCAM1) and thyroid hormone receptor alpha chain (THRA) genes to ovine chromosomes 3 and 11.

Affinity, Kinetics, and Thermodynamics of E-selectin Binding to E-selectin Ligand-1

E-selectin is an endothelial adhesion molecule, which mediates the tethering and rolling of leukocytes on vascular endothelium. It recognizes the glycoprotein E-selectin ligand-1 (ESL-1) as a major binding partner on mouse myeloid cells. Using surface plasmon resonance, we measured the kinetics and affinity of binding of monomeric E-selectin to ESL-1 isolated from mouse bone marrow cells. E-selectin bound to ESL-1 with a fast dissociation rate constant of 4.6 s(-1) and a calculated association rate constant of 7.4 x 10(4) m(-1) s(-1). We determined a dissociation constant (K(d)) of 62 microm, which resembles the affinity of L-selectin binding to glycosylation-dependent cell adhesion molecule-1. The affinity of the E-selectin-ESL-1 interaction did not change significantly when the temperature was varied from 5 degrees C to 37 degrees C, indicating that the enthalpic contribution to the binding is small at physiological temperatures, and that, in contrast to typical protein-carbohydrate interactions, binding is driven primarily by favorable entropic changes. Interestingly, surface plasmon resonance experiments with recombinant ESL-1 from alpha 1,3-fucosyltransferase IV-expressing Chinese hamster ovary cells showed a very similar K(d) of 66 microm, suggesting that this fucosyltransferase is sufficient to produce fully functional recombinant ESL-1. Following the recent description of the affinity and kinetics of the selectin-ligand pairs L-selectin-glycosylation-dependent cell adhesion molecule-1 and P-selectin-P-selectin glycoprotein ligand-1, this is the first determination of the parameters of E-selectin binding to one of its naturally occurring ligands.

Regulation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) gene in the mouse mammary gland differs from that of casein genes

Mouse glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), also known as mC26 and homologous to bovine PP3, is a milk protein synthesized in the mammary gland. Several studies have investigated the regulation of casein, the major milk protein, gene in the mammary gland, but little is known about GlyCAM-1. Here we examined GlyCAM-1 gene expression in mouse mammary epithelial cells. First, we detected GlyCAM-1 expression in mammary epithelial cells in situ by immunohistochemistry; almost all mammary epithelial cells of the lactating mouse expressed GlyCAM-1. Second, mammary epithelial cells were digested with collagenase and cultured with insulin, prolactin and/or glucocorticoid. α-Casein and β-casein genes were expressed following treatment with insulin, prolactin and glucocorticoid. In contrast, GlyCAM-1 expression could not be detected with any combination of these three hormones. We also analyzed changes in the levels of GlyCAM-1 and caseins mRNAs in cultured cells. The addition of hormones to the culture medium increased casein mRNAs, but surprisingly reduced GlyCAM-1 mRNA. Our results suggest that the mechanisms that regulate GlyCAM-1 gene in mammary cells of lactating mice are different from those involved in the regulation of casein genes.

The 2-Naphthylmethyl (NAP) Group in Carbohydrate Synthesis: First Total Synthesis of the GlyCAM-1 Oligosaccharide Structures

Total syntheses of the GlyCAM-1 (glycosylation-dependent cell adhesion molecule-1) oligosaccharide structures: {α-NeuAc-(2→3)-β-Gal-(1→4)-[α-Fuc-(1→3)]-β-(6-O-SO3Na)-GlcNAc-(1→6)}-[α-NeuAc-(2→3)-β-Gal-(1→3)]-α-GalNAc-OMe (1) and {α-NeuAc-(2→3)-β-Gal-(1→4)-[α-Fuc-(1→3)]-β-GlcNAc-(1→6)}-[α-NeuAc-(2→3)-β-Gal-(1→3)]-α-GalNAc-OMe (2) through a novel sialyl Lewisx tetrasaccharide donor are described. Employing sequential glycosylation strategy, the starting trisaccharide was regio- and stereoselectively constructed through coupling of a disaccharide imidate with the monosaccharide acceptor phenyl-6-O-naphthylmethyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside with TMSOTf as a catalyst without affecting the SPh group. The novel sialyl Lewisx tetrasaccharide donor 3 was then obtained by α-L-fucosylation of trisaccharide acceptor with the 2,3,4-tri-O-benzyl-1-thio-β-L-fucoside donor. The structure of the novel sialyl Lewisx tetrasaccharide was established by a combination of 2D DQF-COSY and 2D ROESY experiments. Target oligosaccharides 1 and 2 were eventually constructed through heptasaccharide which was obtained by regioselective assembly of advanced sialyl Lewisx tetrasaccharide donor 3 and a sialylated trisaccharide acceptor in a predictable and controlled manner. Finally, target heptasaccharides 1 and 2 were fully characterized by 2D DQF-COSY, 2D ROESY, HSQC, HMBC experiments and FAB mass spectroscopy.

The 2-naphthylmethyl (NAP) group in carbohydrate synthesis: first total synthesis of the GlyCAM-1 oligosaccharide structures.

Total syntheses of the GlyCAM-1 (glycosylation-dependent cell adhesion molecule-1) oligosaccharide structures: [alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 4)-[alpha-Fuc-(1 --> 3)]-beta-(6-O-SO3Na)-GlcNAc-(1 --> 6)]-[alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 3)]-alpha-GalNAc-OMe (1) and [alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 4)-[alpha-Fuc-(1 --> 3)]-beta-GlcNAc-(1 --> 6)]-[alpha-NeuAc-(2 3)-beta-Gal-(1 --> 3)]-alpha-GalNAc-OMe (2) through a novel sialyl LewisX tetrasaccharide donor are described. Employing sequential glycosylation strategy, the starting trisaccharide was regio- and stereoselectively constructed through coupling of a disaccharide imidate with the monosaccharide acceptor phenyl-6-O-naphthylmethyl-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside with TMSOTf as a catalyst without affecting the SPh group. The novel sialyl Lewisx tetrasaccharide donor 3 was then obtained by alpha-L-fucosylation of trisaccharide acceptor with the 2,3,4-tri-O-benzyl-1-thio-beta-L-fucoside donor. The structure of the novel sialyl Lewisx tetrasaccharide was established by a combination of 2D DQF-COSY and 2D ROESY experiments. Target oligosaccharides 1 and 2 were eventually constructed through heptasaccharide which was obtained by regioselective assembly of advanced sialyl Lewisx tetrasaccharide donor 3 and a sialylated trisaccharide acceptor in a predictable and controlled manner. Finally, target heptasaccharides 1 and 2 were fully characterized by 2D DQF-COSY, 2D ROESY, HSQC, HMBC experiments and FAB mass spectroscopy.

Glycosylation-Dependent Cell Adhesion Molecule 1 (GlyCAM 1) Is Induced by Prolactin and Suppressed by Progesterone in Mammary Epithelium

Glycosylation-Dependent Cell Adhesion Molecule 1 (GlyCAM 1) Is Induced by Prolactin and Suppressed by Progesterone in Mammary Epithelium

Glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1) is induced by prolactin and suppressed by progesterone in mammary epithelium.

Glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1), a mucin-like endothelial glycoprotein, was induced by PRL and suppressed by progesterone in the mammary gland of mice, and in HC11 mouse mammary epithelial cells. Complementary DNA microarray analysis revealed that expression of GlyCAM 1 was reduced in the mammary gland of PRL-gene disrupted mice (PRL-/-) compared with control (PRL+/-) littermates. This result was confirmed by in situ hybridization and immunostaining. The messenger RNA (mRNA) encoding GlyCAM 1 was present in mammary epithelia of PRL-stimulated mice. Immunohistochemistry indicated that GlyCAM 1 protein was detectable both in mammary epithelia and in the ductal lumen in PRL+/- virgin mice, but not in PRL-/- mice. GlyCAM 1 mRNA was highly induced by grafting pituitary glands from normal littermates. Trace amounts of mRNA for GlyCAM 1 were detected by RT-PCR in mammary tissue of PRL-/- mice. Progesterone inhibited both basal and PRL-stimulated GlyCAM 1 transcription. In HC11 cells, GlyCAM 1 mRNA was induced in cells treated with insulin, dexamethasone, and PRL. Similar to the in vivo studies, progesterone inhibited the induction of GlyCAM 1 transcription. In CHO cells, PRL stimulated transcription of a luciferase reporter gene containing an 800-bp promoter fragment of GlyCAM 1, and progesterone partially suppressed the PRL effect. These data demonstrate that expression of GlyCAM 1 in mammary gland is under the control of both PRL and progesterone.

Lymphocyte homing to allografts.

A complex system of lymphocyte trafficking has evolved to provide immune surveillance and recognition of foreign antigens (1). When the regulation of this process is disrupted leukocyte recruitment continues inappropriately resulting in pathological inflammation (2, 3). During graft rejection activa

Camel (camelus dromedarius) milk PP3: evidence for an insertion in the amino-terminal sequence of the camel milk whey protein.

The camel (camelus dromedarius) milk proteose peptone 3 (PP3) was purified successively by size exclusion fast protein liquid chromatography and reversed phase high performance liquid chromatography and then characterized by amino acid residue composition determination and chemical microsequencing after CNBr or trypsin cleavages. In comparison with the previously reported structure of camel milk whey protein, the camel PP3 contains an insertion in the N-terminal region which has approximately 24 residues, whereas the remaining C-terminal regions of these two homologous proteins are essentially identical. The camel PP3 seems to contain a potential O-glycosylation site localized in this insertion and 2 or 3 phosphorylated serine residues. PP3 belongs to the glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) family and could therefore play an immunological role in the camel or its suckling young.

Sulfation of a high endothelial venule-expressed ligand for L-selectin. Effects on tethering and rolling of lymphocytes.

During lymphocyte homing, L-selectin mediates the tethering and rolling of lymphocytes on high endothelial venules (HEVs) in secondary lymphoid organs. The L-selectin ligands on HEV are a set of mucin-like glycoproteins, for which glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) is a candidate. Optimal binding in equilibrium measurements requires sulfation, sialylation, and fucosylation of ligands. Analysis of GlyCAM-1 has revealed two sulfation modifications (galactose [Gal]-6-sulfate and N-acetylglucosamine [GlcNAc]-6-sulfate) of sialyl Lewis x. Recently, three related sulfotransferases (keratan sulfate galactose-6-sulfotransferase [KSGal6ST], high endothelial cell N-acetylglucosamine-6-sulfotransferase [GlcNAc6ST], and human GlcNAc6ST) were cloned, which can generate Gal-6-sulfate and GlcNAc-6-sulfate in GlyCAM-1. Imparting these modifications to GlyCAM-1, together with appropriate fucosylation, yields enhanced rolling ligands for both peripheral blood lymphocytes and Jurkat cells in flow chamber assays as compared with those generated with exogenous fucosyltransferase. Either sulfation modification results in an increased number of tethered and rolling lymphocytes, a reduction in overall rolling velocity associated with more frequent pausing of the cells, and an enhanced resistance of rolling cells to detachment by shear. All of these effects are predicted to promote the overall efficiency of lymphocyte homing. In contrast, the rolling interactions of E-selectin transfectants with the same ligands are not affected by sulfation.

Alternative splicing of lactophorin mRNA from lactating mammary gland of the camel (Camelus dromedarius).

The objective of this study was to determine the corrected structure of lactophorin, a major whey protein in camel milk. The protein had 60.4% amino acid sequence identity to a proteose peptone component 3 protein from bovine whey and 30.3% identity to the glycosylation-dependent cell adhesion molecule 1 in mice. The N-terminal heterogeneity of the protein was a result of alternative mRNA splicing. About 75% of the protein was expressed as a long variant A with 137 amino acid residues and a molecular mass of 15.7 kDa; about 25% was as a short variant B with 122 amino acid residues and a molecular mass of 13.8 kDa. Both proteins are probably threefold phosphorylated. In contrast to the related proteins, no glycosylation was found in camel lactophorin. Because of this difference, specific interaction with carbohydrate binding proteins, as reported for the murine protein, can be excluded, and a function of the protein other than cell recognition or rotaviral inhibition is proposed. The concentration of lactophorin in camel milk was found to be about three times higher than the concentration of the bovine homologue in bovine milk. Pronounced similarities existed between the primary and secondary structures of bovine and camel proteins. We speculated that camel lactophorin has a similar function to that of bovine protein in milk, which is supposed to be the prevention of fat globule aggregation and the inhibition of spontaneous lipolysis by lipoprotein lipase.