Determination of disulfide bond assignments and N-glycosylation sites of the human gastrointestinal carcinoma antigen GA733-2 (CO17-1A, EGP, KS1-4, KSA, and Ep-CAM)

The GA733-2 antigen is a cell surface glycoprotein highly expressed on most human gastrointestinal carcinoma and at a lower level on most normal epithelia. It is an unusual cell-cell adhesion protein that does not exhibit any obvious relationship to the four known classes of adhesion molecules. In this study, the disulfide-bonding pattern of the GA733-2 antigen was determined using matrix-assisted laser desorption/ionization mass spectrometry and N-terminal sequencing of purified tryptic peptides treated with 2-[2'-nitrophenylsulfonyl]-3-methyl-3-bromoindolenine or partially reduced and alkylated. Numbering GA733-2 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys4, Cys2-Cys6, and Cys3-Cys5, which is a novel pattern for a cysteine-rich domain instead of the expected epidermal growth factor-like disulfide structure. The next three disulfide linkages are Cys7-Cys8, Cys9-Cys10, and Cys11-Cys12, consistent with the recently determined disulfide pattern of the thyroglobulin type 1A domain of insulin-like growth factor-binding proteins 1 and 6. Analysis of glycosylation sites showed that GA733-2 antigen contained N-linked carbohydrate but that no O-linked carbohydrate groups were detected. Of the three potential N-linked glycosylation sites, Asn175 was not glycosylated, whereas Asn88 was completely glycosylated, and Asn51 was partially glycosylated. These data show that the extracellular domain of the GA733-2 antigen consists of three distinct domains; a novel cysteine-rich N-terminal domain (GA733 type 1 motif), a cysteine-rich thyroglobulin type 1A domain (GA733 type 2 motif), and a unique nonglycosylated domain without cysteines (GA733 type 3 motif).     

N-linked glycosylation regulates human proteinase-activated receptor-1 cell surface expression and disarming via neutrophil proteinases and thermolysin

Proteinase-activated receptor 1 (PAR(1)) induces activation of platelet and vascular cells after proteolytic cleavage of its extracellular N terminus by thrombin. In pathological situations, other proteinases may be generated in the circulation and might modify the responses of PAR(1) by cleaving extracellular domains. In this study, epitope-tagged wild-type human PAR(1) (hPAR(1)) and a panel of N-linked glycosylation-deficient mutant receptors were permanently expressed in epithelial cells (Kirsten murine sarcoma virus-transformed rat kidney cells and CHO cells). We have analyzed the role of N-linked glycosylation in regulating proteinase activation/disarming and cell global expression of hPAR(1). We reported for the first time that glycosylation in the N terminus of hPAR(1) downstream of the tethered ligand (especially Asn(75)) governs receptor disarming to trypsin, thermolysin, and the neutrophil proteinases elastase and proteinase 3 but not cathepsin G. In addition, hPAR(1) is heavily N-linked glycosylated and sialylated in epithelial cell lines, and glycosylation occurs at all five consensus sites, namely, Asn(35), Asn(62), Asn(75), Asn(250), and Asn(259). Removing these N-linked glycosylation sequons affected hPAR(1) cell surface expression to varying degrees, and N-linked glycosylation at extracellular loop 2 (especially Asn(250)) of hPAR(1) is essential for optimal receptor cell surface expression and receptor stability.     

Identification of two N-linked glycosylation sites within the core of the simian immunodeficiency virus glycoprotein whose removal enhances sensitivity to soluble CD4

Using PCR mutagenesis to disrupt the NXT/S N-linked glycosylation motif of the Env protein, we created 27 mutants lacking 1 to 5 of 14 N-linked glycosylation sites within regions of gp120 lying outside of variable loops 1 to 4 within simian immunodeficiency virus strain 239 (SIV239). Of 18 mutants missing N-linked glycosylation sites predicted to lie within 10 A of CD4 contact sites, the infectivity of 12 was sufficient to measure sensitivity to neutralization by soluble CD4 (sCD4), pooled immune sera from SIV239-infected rhesus macaques, and monoclonal antibodies known to neutralize certain derivatives of SIV239. Three of these 12 mutants (g3, lacking the 3rd glycan at position 79; g11, lacking the 11th glycan at position 212; and g3,11, lacking both the 3rd and 11th glycans) were approximately five times more sensitive to neutralization by sCD4 than wild-type (WT) SIV239. However, these same mutants were no more sensitive to neutralization than WT by pooled immune sera. The other 9 of 12 replication-competent mutants in this group were no more sensitive to neutralization than the WT by any of the neutralizing reagents. Six of the nine mutants that did not replicate appreciably had three or more glycosylation sites eliminated; the other three replication-deficient strains involved mutation of site 15. Our results suggest that elimination of glycan attachment sites 3 and 11 enhanced the exposure of contact residues for CD4. Thus, glycans at positions 3 and 11 of SIV239 gp120 may be particularly important for shielding the CD4-binding site from antibody recognition.     

Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked carbohydrate chains

Organic anion transport protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous transport proteins, is expressed on the basolateral (sinusoidal) surface of rat hepatocytes. The organization of oatp1a1 within the plasma membrane has not been well defined, and computer-based models have predicted possible 12- as well as 10-transmembrane domain structures. Which of oatp1a1's four potential N-linked glycosylation sites are actually glycosylated and their influence on transport function have not been investigated in a mammalian system. In the present study, topology of oatp1a1 in the rat hepatocyte plasma membrane was examined by immunofluorescence analysis using an epitope-specific antibody designed to differentiate a 10- from a 12-transmembrane domain model. To map glycosylation sites, the asparagines at the each of the four N-linked glycosylation consensus sites were mutagenized to glutamines. Mutagenized oatp1a1 constructs were expressed in HeLa cells, and effects on protein expression and transport activity were assessed. These studies revealed that oatp1a1 is a 12-transmembrane-domain protein in which the second and fifth extracellular loops are glycosylated at asparagines 124, 135, and 492, whereas the potential glycosylation site at asparagine 62 is not utilized, consistent with its position in a transmembrane domain. Constructs in which more than one glycosylation site were eliminated had reduced transport activity but not necessarily reduced transporter expression. This was in accord with the finding that fully unglycosylated oatp1a1 was well expressed but located intracellularly with limited transport ability as a consequence of its reduced cell surface expression.     

Determination of carbohydrate structures N-linked to soluble CD154 and characterization of the interactions of CD40 with CD154 expressed in Pichia pastoris and Chinese hamster ovary cells.

CD40-CD154 (CD40 ligand) interactions are essential for the development of protective immunity. Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154. However, these studies have not examined the structure or biological function of the carbohydrate on CD154. Human CD154 contains a single N-linked glycosylation site at asparagine 240. We have characterized the interactions between CD40 and soluble (s) CD154 in which sCD154 contains different types of carbohydrates. Detailed carbohydrate analysis revealed high-mannose structures on sCD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates. sCD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10-30 nM, and it functionally induced CD69 and CD95 expression on primary B cells. Together, these results indicate that the presence of varied types of N-linked glycans on asparagine 240 of CD154 does not play a significant role in the CD40-CD154 interactions.     

Production and characterization of human soluble CD83 fused with the fragment crystallizable region of human IgG1 in Pichia pastoris

The cell surface protein CD83 belongs to the immunoglobulin superfamily and is highly expressed on mature dendritic cells. The soluble form of CD83, sCD83, is a potential immune suppressor. In a previous study, recombinant soluble CD83 was expressed in Escherichia coli, resulting in a lack of functional glycosylation. Although eukaryotic cell systems for producing sCD83 offer the advantages of protein processing, folding, and posttranslational modification, these systems are complicated, expensive, and produce low levels of protein. To obtain more efficient expression of sCD83, we expressed human sCD83 fused with fragment crystallizable region of human IgG1 (hIgG1 Fc) in Pichia pastoris. Under the optimal conditions (time of induction, 48 h; inoculum density (OD₆₀₀), 80; concentration of methanol, 3.0 %; pH 7.0–8.0; concentration of casamino acid, 5.0 %), the purified human sCD83-hIgG1 Fc (hsCD83-Ig) fusion protein existed as dimers at 25–30 mg/L culture. Treatment with PNGase F showed that purified hsCD83-Ig was modified by N-linked glycosylation. Moreover, the hsCD83-Ig expressed in the P. pastoris system could suppress lymphocyte proliferation in ConA-stimulated and one-way mixed lymphocyte reaction systems. Thus, hsCD83-Ig expressed in P. pastoris is functional and may be used in experimental therapies for graft rejection, graft-versus-host disease, and autoimmune diseases.     

Expression of human glycophorin A in wild type and glycosylation-deficient Chinese hamster ovary cells. Role of N- and O-linked glycosylation in cell surface expression.

Glycophorin A, the most abundant sialoglycoprotein on human red blood cells, carries several medically important blood group antigens. To study the role of glycosylation in surface expression and antigenicity of this highly glycosylated protein (1 N-linked and 15 O-linked oligosaccharides), glycophorin A cDNA (M-allele) was expressed in Chinese hamster ovary (CHO) cells. Both wild type CHO cells and mutant CHO cells with well defined glycosylation defects were used. Glycophorin A was well expressed on the surface of transfected wild type CHO cells. On immunoblots, the CHO cells expressed monomer (approximately 38 kDa) and dimer forms of glycophorin A which co-migrated with human red blood cell glycophorin A. The transfected cells specifically expressed the M blood group antigen when tested with mouse monoclonal antibodies. Tunicamycin treatment of these CHO cells did not block surface expression of glycophorin A, indicating that, in the presence of normal O-linked glycosylation, the N-linked oligosaccharide is not required for surface expression. To study O-linked glycosylation, glycophorin A cDNA was transfected into the Lec 2, Lec 8, and ldlD glycosylation-deficient CHO cell lines. Glycophorin A with truncated O-linked oligosaccharides was well expressed on the surface of ldlD cells (cultured in the presence of N-acetylgalactosamine alone), Lec 2 cells, and Lec 8 cells with monomers of approximately 25 kDa, approximately 33 kDa, and approximately 25 kDa, respectively. In contrast, non-O-glycosylated glycophorin A (approximately 19-kDa monomers) was poorly expressed on the surface of ldlD cells cultured in the absence of both galactose and N-acetylgalactosamine. Thus, under these conditions, in the absence of O-linked glycosylation, the N-linked oligosaccharide itself is not able to support appropriate surface expression of glycophorin A in transfected CHO cells.     

Glycosylation profiles of the human colorectal cancer A33 antigen naturally expressed in the human colorectal cancer cell line SW1222 and expressed as recombinant protein in different insect cell lines

The A33 antigen is a cell surface glycoprotein expressed in human gastrointestinal epithelium and in 95% of colorectal cancers. We have compared the N-linked glycosylation profile of A33 antigen naturally expressed in a human colorectal cancer cell line with recombinant human A33 antigen (rA33) produced in insect cell culture using the baculovirus expression vector. N-Linked glycans were enzymatically released from the protein, and glycan composition was analyzed by HPLC. In three insect cell lines tested (Sf-21, Tn5B1-4, and Tn-4s), glycosylation of rA33 was dominated by high mannose structures (M5Gn2 to M9Gn2; 78-95% of total N-linked glycans), with M8Gn2 being the single most abundant glycoform. A33 antigen naturally expressed in the SW1222 human colon cancer cell line (A33) also possessed a high abundance of high mannose glycans (72%). No complex glycosylation was detected on rA33 expressed in insect cells. Natural A33 was galactosylated to a small extent (6%). These results illustrate a case of similar glycosylation of a glycoprotein between a recombinant version produced in insect cell culture and its counterpart naturally expressed in human cell culture.     

Molecular Modeling of Immunoglobulin Superfamily Proteins: Predicting the Three Dimensional Structure of the Extracellular Domain of CTLA-4 (CD152)

The interactions between CD28/CTLA-4 (CD152) on T cells and their ligands CD80/CD86 on antigen presenting cells provide costimulatory signals critical for T cell activation. CD28/CTLA-4 and CD80/CD86 are members of the immunoglobulin superfamily (IgSF). CD28 and CTLA-4 both contain a single extracellular immunoglobulin (Ig) domain which binds CD80/CD86. Here we report modeling studies on the three-dimensional (3D) structure of the CTLA-4 binding domain. Since CTLA-4 displays only very weak sequence homology to proteins with known 3D structure, conventional modeling techniques were difficult to apply. Structure-oriented sequence comparison, consensus residue analysis, conformational searching, and inverse folding calculations were employed to aid in the generation of a comparative CTLA-4 model. Regions of high and low prediction confidence were identified, and the sequence-structure compatibility of the model was determined. Characteristics of the modeled structure, which resembles an Ig V domain, were analyzed, and the model was used to map N-linked glycosylation sites and residues critical for CTLA-4 function. The modeling approach described here can be applied to predict 3D structures of other IgSF proteins.     

A Molecular Model of Inducible Costimulator Protein and Three-Dimensional Analysis of its Relation to the CD28 Family of T Cell-Specific Costimulatory Receptors

Inducible costimulator protein (ICOS) has recently been identified as a new member of the CD28 family of T cell costimulatory molecules. A molecular model of the extracellular immunoglobulin-like domain of ICOS was built based on the structure of CD152, another member of the CD28 family. Despite low sequence identity, ICOS shares consensus residues characteristic of immunoglobulin variable-type domains with CD152 and CD28 and also some unique features, suggesting that their three-dimensional structures are more similar to each other than to other proteins belonging to the immunoglobulin superfamily. The ICOS model was used to study sequence conservation in three dimensions and to compare the distribution of N-linked glycosylation sites in the extended CD28 family. The limited number of residues outside consensus/core positions that are conserved in ICOS and CD28 and/or CD152 are widely distributed over the extracellular domain. A few residues in CD152 and CD28 that are critical for binding of CD80/CD86 are also conserved in ICOS. However, the region in ICOS that corresponds to the CD80/CD86 binding site is masked by N-linked glycosylation. This suggests that this site is not available for binding of CD80/CD86 or other ligands. ICOS has probably diverged early from CD28 and CD152 and developed the capacity to recognize ligand(s) other than CD80/CD86, very likely utilizing a different molecular region and mechanism for binding.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s008940050116     

Molecular cloning and characterization of a novel murine T cell surface antigen, YE1/48

YE1/48 is a murine cell surface disulphide-linked dimeric Ag consisting of two 45,000-50,000 Mr subunits. It is expressed on some T lymphoma lines at high levels but its expression on normal lymphocytes is very low. The functional significance of this Ag is currently unknown. We have now cloned a cDNA encoding the YE1/48. Sequence analysis revealed that it encodes a Type II membrane protein of 262 amino acids (30,500 MW), with 44 amino acids in the N-terminal cytoplasmic domain, 22 amino acids in the transmembrane domain and 196 amino acids in the C-terminal extracellular domain. There are three potential N-linked glycosylation sites in the extracellular domain all of which are probably used in the mature protein. No significant homology can be identified with other known protein sequences in the data base or with human CD28(T44), a human T cell activation antigen consisting of two 44,000 Mr subunits. The protein sequence includes in its extracellular domain the arginine-glycine-aspartic acid tripeptide, a potential cell-adhesive binding site, and a sequence similar to the consensus domain of any metal-binding proteins. However, whether these sequences are functional is unknown. Genomic Southern analysis of C57BL/6, BALB/c and C3H mice has demonstrated a restriction fragment length polymorphism. The analysis has also strongly suggested the existence of some other genes with sequences highly homologous to the YE1/48 gene. The YE1/48 gene appears to be expressed at very low levels in a wide range of lymphoid cells with no restriction to their differentiation stages. Interestingly, YE1/48 expression appears to be induced in pre-B cells after transformation by Abelson virus, suggesting an association of YE1/48 expression with the transformation of T and pre-B Cells.     

Affinity and kinetic analysis of the interaction of the cell adhesion molecules rat CD2 and CD48.

CD2 is a plasma membrane glycoprotein present on T lymphocytes that functions as a cell adhesion molecule (CAM). The CD2 counter-receptor in rodents is the structurally-related CAM CD48. Intercellular adhesion involves the formation of multiple CAM complexes between adhering cells and de-adhesion requires disruption of these complexes. To gain an insight into the initiation and termination of intercellular adhesion, the kinetics and affinity of the rat CD2-CD48 interaction was analysed using a BIAcore instrument, which enables the monitoring of protein binding in real time. A soluble chimeric protein, comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4), bound to immobilized soluble CD2 (sCD2) with a KD of 90 microM. The affinity was also determined in the reverse orientation and sCD2 was shown to bind immobilized sCD48-CD4 with a comparable KD of 60 microM. sCD48-CD4 bound to immobilized deglycosylated sCD2 with a KD of 125 microM, indicating that glycosylation of sCD2 has little effect on the affinity of the interaction. The low affinity was the result of an extremely rapid off-rate constant (K(off) > or = 6 s-1), whereas the on-rate constant was unremarkable (K(on) > or = 10(5) M-1s-1). The kinetic analysis revealed that small amounts of multimeric aggregates of sCD48-CD4 formed in concentrated preparations. Our experience suggests that even low concentrations (< 2%) of these aggregates may be a cause of artifactually high affinity values when analysing low-affinity protein interactions. In conclusion, this study provides the first detailed analysis of the kinetics and affinity of monomeric CAM interactions and suggests that binding between CAMs may be weaker than anticipated.     

Endotoxin/lipopolysaccharide activates NF-kappa B and enhances tumor cell adhesion and invasion through a beta 1 integrin-dependent mechanism

Beta(1) integrins play a crucial role in supporting tumor cell attachment to and invasion into the extracellular matrix. Endotoxin/LPS introduced by surgery has been shown to enhance tumor metastasis in a murine model. Here we show the direct effect of LPS on tumor cell adhesion and invasion in extracellular matrix proteins through a beta(1) integrin-dependent pathway. The human colorectal tumor cell lines SW480 and SW620 constitutively expressed high levels of the beta(1) subunit, whereas various low levels of alpha(1), alpha(2), alpha(4), and alpha(6) expression were detected. SW480 and SW620 did not express membrane-bound CD14; however, LPS in the presence of soluble CD14 (sCD14) significantly up-regulated beta(1) integrin expression; enhanced tumor cell attachment to fibronectin, collagen I, and laminin; and strongly promoted tumor cell invasion through the Matrigel. Anti-beta(1) blocking mAbs (4B4 and 6S6) abrogated LPS- plus sCD14-induced tumor cell adhesion and invasion. Furthermore, LPS, when combined with sCD14, resulted in NF-kappaB activation in both SW480 and SW620 cells. Inhibition of the NF-kappaB pathway significantly attenuated LPS-induced up-regulation of beta(1) integrin expression and prevented tumor cell adhesion and invasion. These results provide direct evidence that although SW480 and SW620 cells do not express membrane-bound CD14, LPS in the presence of sCD14 can activate NF-kappaB, up-regulate beta(1) integrin expression, and subsequently promote tumor cell adhesion and invasion. Moreover, LPS-induced tumor cell attachment to and invasion through extracellular matrix proteins is beta(1) subunit-dependent.     

Extracellular domain nicotinic acetylcholine receptors formed by alpha4 and beta2 subunits

Models of the extracellular ligand-binding domain of nicotinic acetylcholine receptors (nAChRs), which are pentameric integral membrane proteins, are attractive for structural studies because they potentially are water-soluble and better candidates for x-ray crystallography and because their smaller size is more amenable for NMR spectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of alpha7 and muscle-type subunits. Specific design requirements leading to high structural fidelity between extracellular domain nAChRs and full-length nAChRs, however, are not well understood. To study these requirements in heteromeric nAChRs, the extracellular domains of alpha4 and beta2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with alpha4beta2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain alpha4beta2 nAChRs formed from subunits with M1 were nearly identical to affinities of alpha4beta2 nAChRs when measured with [3H]epibatidine, cytisine, nicotine, and acetylcholine. Velocity sedimentation suggested that these extracellular domain nAChRs predominantly formed pentamers. The yield of these extracellular domain nAChRs was about half the yield of alpha4beta2 nAChRs. In contrast, [3H]epibatidine binding was not detected from the extracellular domain alpha4 and beta2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both alpha4 and beta2 play an important role for efficient expression of extracellular domain alpha4beta2 nAChRs that are high fidelity structural models of full-length alpha4beta2 nAChRs.     

Role of CD3 gamma in T cell receptor assembly

The T cell receptor (TCR) consists of the Ti alpha beta heterodimer and the associated CD3 gamma delta epsilon and zeta 2 chains. The structural relationships between the subunits of the TCR complex are still not fully known. In this study we examined the role of the extracellular (EC), transmembrane (TM), and cytoplasmic (CY) domain of CD3 gamma in assembly and cell surface expression of the complete TCR in human T cells. A computer model indicated that the EC domain of CD3 gamma folds as an Ig domain. Based on this model and on alignment studies, two potential interaction sites were predicted in the EC domain of CD3 gamma. Site-directed mutagenesis demonstrated that these sites play a crucial role in TCR assembly probably by binding to CD3 epsilon. Mutagenesis of N-linked glycosylation sites showed that glycosylation of CD3 gamma is not required for TCR assembly and expression. In contrast, treatment of T cells with tunicamycin suggested that N-linked glycosylation of CD3 delta is required for TCR assembly. Site-directed mutagenesis of the acidic amino acid in the TM domain of CD3 gamma demonstrated that this residue is involved in TCR assembly probably by binding to Ti beta. Deletion of the entire CY domain of CD3 gamma did not prevent assembly and expression of the TCR. In conclusion, this study demonstrated that specific TCR interaction sites exist in both the EC and TM domain of CD3 gamma. Furthermore, the study indicated that, in contrast to CD3 gamma, glycosylation of CD3 delta is required for TCR assembly and expression.     

Mutational analysis of N-glycosylation recognition sites on the biochemical properties of Aspergillus kawachii alpha-L-arabinofuranosidase 54

A role for N-linked oligosaccharides on the biochemical properties of recombinant alpha-l-arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N-linked glycosylation motifs (Asn(83)-Thr-Thr and Asn(202)-Ser-Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N-linked glycosylation sites are located in the catalytic domain. Asn(83), Asn(202), and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn(202) may contribute to thermostability and catalysis.     

Functional role of N-linked glycosylation on the rat melanin-concentrating hormone receptor 1

Melanin-concentrating hormone (MCH) is known to act through two G-protein-coupled receptors MCHR1 and MCHR2. MCHR1 has three potential sites (Asn13, Asn16 and Asn23) for N-linked glycosylation in its extracellular amino-terminus which may modulate its reactivity. Site-directed mutagenesis of the rat MCHR1 cDNA at single or multiple combinations of the three potential glycosylation sites was used to examine the role of the putative carbohydrate chains on receptor activity. It was found that all three potential N-linked glycosylation sites in MCHR1 were glycosylated, and that N-linked glycosylation of Asn23 was necessary for full activity. Furthermore, disruption of all three glycosylation sites impaired proper expression at the cell surface and receptor activity. These data outline the importance of the N-linked glycosylation of the MCHR1.     

Variations in the cytoskeletal interaction and posttranslational modification of the CD44 homing receptor in macrophages

Murine CD44 is a cell surface glycoprotein that is thought to play a role in leukocyte migration. We studied the structure and expression of CD44 on two populations of macrophages: those that reside in the peritoneum of unprimed mice, and those that have been elicited to migrate into the peritoneum by the intraperitoneal injection of agents that cause localized inflammatory responses. Our studies reveal structural variations in both the extracellular and intracellular domains of CD44 expressed by these two macrophage populations. The form of CD44 in elicited macrophages has an apparent molecular mass that is approximately 5 kD greater and more heterogenous than that in resident macrophages. This structural changes is posttranslational, extracellular, and apparently reflects increases in N-linked glycosylation. It is also specific for CD44 and does not occur with several other glycoproteins examined. This novel regulation of glycosylation may play an important role in the ability of CD44 to bind to different substrates, particularly lectin-like ligands. In addition, we demonstrate that CD44 in resident macrophages is divided into two pools, one containing nonphosphorylated, cytoskeletally associated CD44, and one containing phosphorylated, unassociated CD44. In contrast, CD44 on the surface of elicited macrophages does not associate with the cytoskeleton. The attachment of CD44 to the cytoskeleton involves either direct or indirect association with actin. The regulated association of CD44 with the cytoskeleton suggests that it may influence or be influenced by macrophage mobility.     

Membrane trafficking of CD98 and its ligand galectin 3 in BeWo cells--implication for placental cell fusion

CD98 heavy chain (CD98hc), expressed at high levels in developing human trophoblasts, is an integral membrane protein with multiple N-linked glycosylation sites and known to be important for cell fusion, adhesion, and amino acid transport. Western blotting and flow cytometry were used to study the effect of brefeldin A, an inhibitor of protein translocation through the Golgi, on CD98hc in the human placental trophoblast cell line BeWo. Although brefeldin A treatment caused increased cell surface expression of CD98hc, a novel partially glycosylated form of the protein was found and, concomitantly, cell fusion was reduced. Western blotting showed that CD98 and galectin 3, a proposed ligand for the glycosylated extracellular domain of CD98hc, co-immunoprecipitated, and double-label immuno-electron microscopy confirmed that CD98hc associated with galectin 3. Furthermore, cell fusion was reduced (specifically) by the disaccharide lactose, a known ligand for the carbohydrate recognition domain of galectin 3, suggesting that the association was functional. Taken together, the data suggest that N-glycosylation of CD98 and subsequent interaction with galectin 3 is critical for aspects of placental cell biology, and provides a rationale for the observation that, in the mouse, truncation of the CD98hc extracellular domain leads to early embryonic lethality [Tsumura H, Suzuki N, Saito H, Kawano M, Otake S, Kozuka Y, Komada H, Tsurudome M & Ito Y (2003) Biochem Biophys Res Commun 308, 847-851].