Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping--a novel approach to assess intermolecular protein contacts

The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3'-dithio-bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping. Comparison of the peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a "head-to-tail" arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-Fab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping (+/- thiol reagent). The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-Fab. The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping (+ thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.     

The increased potency of cross-linked lymphocyte function-associated antigen-3 (LFA-3) multimers is a direct consequence of changes in valency.

We have used chemically cross-linked dimers, trimers, and tetramers of lymphocyte function-associated antigen-3 (LFA-3) to study the role of multivalency in the interaction of the protein with its receptor, CD2. The cross-linked adducts showed enhanced activity in systems where LFA-3 has been shown to (i) block LFA-3/CD2 interactions in a rosetting assay and (ii) provide through the CD2 on peripheral blood lymphocytes a trigger for T-cell proliferation. The level of increase was directly related to the valency state of the multimers. In the rosetting assay, the dimers, trimers, and tetramers, by weight, exhibited 15-, 150-, and 430-fold increases in activity over monomeric LFA-3. In the proliferation assay, the tetramer produced a 6-fold increase in thymidine incorporation at 0.06 micrograms/ml, the trimer was 100 times less active than the tetramer, and the dimer and monomer were inactive. The LFA-3 multimers were generated using a three-step cross-linking chemistry that was targeted at the carbohydrates on LFA-3. With this procedure over 60% of the starting protein was converted into multimers with no effect on function. The cross-linking approach should be applicable to other surface antigens, providing a simple method for analyzing multivalent interactions.     

Three distinct actin-attached structural states of myosin in muscle fibers

We have used thiol cross-linking and electron paramagnetic resonance (EPR) to resolve structural transitions of myosin's light chain domain (LCD) and catalytic domain (CD) that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain for endogenous regulatory light chain, with full retention of function. To trap myosin in a structural state analogous to the elusive posthydrolysis ternary complex A.M'.D.P, we used pPDM to cross-link SH1 (Cys(707)) to SH2 (Cys(697)) on the CD. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), SH1-SH2 cross-linked (A.M'.D.P analog), and rigor (A.M.D). EPR showed that the LCD of cross-linked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to cross-link SH1-SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 cross-linking traps a state in which both the CD and LCD are intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the cross-linked state is an A.M'D.P analog on the force generation pathway.     

Potent inhibition of CD4/TCR-mediated T cell apoptosis by a CD4-binding glycoprotein secreted from breast tumor and seminal vesicle cells

We previously isolated a CD4 ligand glycoprotein, gp17, from human seminal plasma; this glycoprotein is identical with gross cystic disease fluid protein-15 (GCDFP-15), a factor specifically secreted from primary and secondary breast tumors. The function of gp17/GCDFP-15 in physiological as well as in pathological conditions has remained elusive thus far. As a follow up to our previous findings that gp17 binds to CD4 with high affinity and interferes with both HIV-1 gp120 binding to CD4 and syncytium formation, we investigated whether gp17 could affect the T lymphocyte apoptosis induced by a separate ligation of CD4 and TCR. We show here that gp17/GCDFP-15 is in fact a strong and specific inhibitor of the T lymphocyte programmed cell death induced by CD4 cross-linking and subsequent TCR activation. The antiapoptotic effect observed in the presence of gp17 correlates with a moderate up-regulation of Bcl-2 expression in treated cells. The presence of gp17 also prevents the down-modulation of Bcl-2 expression in Bcl-2bright CD4+ T cells that is caused by the triggering of apoptosis. Our results suggest that gp17 may represent a new immunomodulatory CD4 binding factor playing a role in host defense against infections and tumors.     

Scorpion-toxin mimics of CD4 in complex with human immunodeficiency virus gp120 crystal structures, molecular mimicry, and neutralization breadth

The binding surface on CD4 for the HIV-1 gp120 envelope glycoprotein has been transplanted previously onto a scorpion-toxin scaffold. Here, we use X-ray crystallography to characterize atomic-level details of gp120 with this transplant, CD4M33. Despite known envelope flexibility, the conformation of gp120 induced by CD4M33 was so similar to that induced by CD4 that localized measures were required to distinguish ligand-induced differences from lattice variation. To investigate relationships between structure, function, and mimicry, an F23 analog of CD4M33 was devised. Structural and thermodynamic analyses showed F23 to be a better molecular mimic of CD4 than CD4M33. F23 also showed increased neutralization breadth, against diverse isolates of HIV-1, HIV-2, and SIVcpz. Our results lend insight into the stability of the CD4 bound conformation of gp120, define measures that quantify molecular mimicry as a function of evolutionary distance, and suggest how such evaluations might be useful in developing mimetic antagonists with increased neutralization breadth.     

Fluorescent labeling of the carbohydrate moieties of human chorionic gonadotropin and alpha 1-acid glycoprotein

A method for covalent attachment of a fluorescent molecule to the carbohydrate moieties of glycoproteins is described. The glycoproteins were oxidized with periodate under mild conditions selective for sialic acid (Van Lenten, L. and Ashwell, G. (1971) J. Biol. Chem. 246, 1889--1894). The resulting aldehydes were condensed with either dansylhydrazine, dansylethylenediamine, or fluoresceinamine followed by reduction with NaCNBH3 and NaBH4. Conjugates prepared with dansylhydrazine were found to be insufficiently stable for spectroscopic analysis, whereas the primary amines produced stable conjugates whose fluorescence polarization (P) was constant for several hours at 37 degrees C. The degree of labeling correlated roughly with the sialic acid contents of the vaious glycoproteins. Very little covalent incorporation was observed with albumin (which is devoid of carbohydrate) or with asialo alpha 1-acid glycoprotein. Exclusion chromatography in the presence of a dissociating agent was sometimes required to remove significant amounts of noncovalently adsorbed dye. Fluorescent-labeled alpha subunits of human chorionic gonadotropin were shown to recombine normally with native beta subunits. However, the labeling procedure appeared to compromise the ability of the beta subunits to recombine. Electrophoretic analysis produced evidence of covalent cross-linking between subunits following periodate oxidation of the intact gonadotropin. The possibility that primary amine groups of the protein compete with added fluorescent amines for reaction with periodate-generated aldehydes is discussed.     

CD44 cross-linking induces integrin-mediated adhesion and transendothelial migration in breast cancer cell line by up-regulation of LFA-1 (alpha L beta2) and VLA-4 (alpha4beta1)

CD44, a widely expressed cell surface glycoprotein, plays a major role in cell-cell adhesion, cell-substrate interaction, lymphocyte homing, and tumor metastasis. For tumor metastasis to occur through the blood vessel and lymphatic vessel pathway, the tumor cells must first adhere to endothelial cells. Recent studies have shown that high expression of CD44 in certain types of tumors is associated with the hematogenic spread of cancer cells. However, the functional relevance of CD44 to tumor cell metastasis remains unknown. In this study, we investigated the mechanisms of CD44 cross-linking-induced adhesion and transendothelial migration of tumor cells using MDA-MB-435S breast cancer cell line. Breast cancer cells were found to express high levels of CD44. Using flow cytometric analysis and immunofluorescence staining, we demonstrated that cross-linking of CD44 resulted in a marked induction of the expression of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) by exocytosis. These results were also observed with the Hs578T breast cancer cell line. Furthermore, LFA-1- and VLA-4-mediated adhesion and transendothelial cancer cell migration were also studied. Anti-LFA-1 mAb or anti-VLA-4 mAb alone had no effect on adhesion or transendothelial cancer cell migration, but were able to inhibit both of these functions when added together. This shows that CD44 cross-linking induces LFA-1 and VLA-4 expression in MDA-MB-435S cells and increases integrin-mediated adhesion to endothelial cells, resulting in the transendothelial migration of breast cancer cells. These observations provide direct evidence of a new function for CD44 that is involved in the induction of LFA-1 and VLA-4 expression by exocytosis in MDA-MB-435S cells. Because these induced integrins promote tumor cell migration into the target tissue, it may be possible to suppress this by pharmacological means, and thus potentially cause a reduction in invasive capability and metastasis.     

CD44v6 promotes proliferation by persisting activation of MAP kinases

CD44v6 is transiently expressed during T cell activation, and constitutively CD44v4-v7 expressing transgenic T cells show accelerated responses towards nominal antigens. The underlying mechanism is unknown. The mouse thymoma EL4 was transfected with CD44 standard isoform (CD44s) or CD44v6 cDNA (EL4-s, EL4-v6). Only EL4-v6 cells proliferated at an over 10-fold higher rate than untransfected cells, displayed up-regulated expression of CD69, CD25, and IL-2, and were protected from apoptosis by CD44v6 cross-linking. In the absence of any stimulus, ERK1/2 was partly phosphorylated, and phosphorylation was significantly increased by CD44v6 cross-linking. The same accounted for JNK, c-jun, and IkappaBalpha. Moreover, NF-kappaB was partly translocated into the nucleus. Instead, CD44s cross-linking induced ERK1/2, JNK, c-jun, and IkappaBalpha phosphorylation only in the context of TCR engagement. No selectively CD44v6 associated transmembrane proteins were uncovered in EL4 cells. However, CD44v6, as opposed to CD44s, did not colocalise with the TCR/CD3 complex after CD3 cross-linking. Furthermore, a CD44-associated 85-kDa protein became hypophosphorylated only after CD44v6 cross-linking. Threonine hypophosphorylation of this protein coincided with the activation of MAP and SAP kinases, which was prohibited in the presence of a phosphatase inhibitor. Thus, CD44v6, distinct to CD44s, stimulates autonomously growth and IL-2 secretion of a thymoma line and rescues cells from apoptosis.     

Lateral membrane protein associations of CD4 in lymphoid cells detected by cross-linking and mass spectrometry

Interactions of membrane proteins are important in various aspects of cell function. However, weak membrane protein-protein interactions are difficult to study using techniques such as co-immunoprecipitations. CD4 is a cell surface protein involved in T cell activation and the binding of the human immunodeficiency virus to HIV target cells. Here we report the use of cross-linking followed by affinity purification of CD4 in combination with mass spectrometry for identification of proteins that are in the proximity of CD4. Besides the components of the CD4 receptor complex, CD4 and lck, we have identified by tandem mass spectrometry 17 tryptic peptides from transferrin receptor CD71, three peptides from protein phosphatase CD45, and one peptide from 4F2 lymphocyte activation antigen CD98. The efficiency of the cross-linking did not correlate with the level of cell surface expression of the detected molecules, excluding a possible bias of the cross-linking toward the most abundant cell surface molecules. Whereas the association of CD4 with CD45 has been reported, the associations with CD71 and CD98 have not been previously described. We used small-scale immunoprecipitation after cross-linking in combination with fluorescence resonance energy transfer (FRET) measurements to investigate the association between CD4 and CD71. Our data show that CD71 self-associates on the cell surface, that a small fraction of CD4 can be detected by copurifying it with CD71 after cross-linking, and that the level of association between CD4 and CD71 significantly increases after phorbol 12-myristate 13-acetate-induced endocytosis of CD4. This suggests that a small fraction of CD4 associates with clusters of CD71. As both molecules undergo endocytic recycling, the association and cross-linking result from their clustering in the same pit and/or vesicle. The CD4-CD98 association probably results from nonspecific cross-linking.     

Insertion of the human immunodeficiency virus CD4 receptor into the envelope of vesicular stomatitis virus particles.

Enveloped virus particles carrying the human immunodeficiency virus (HIV) CD4 receptor may potentially be employed in a targeted antiviral approach. The mechanisms for efficient insertion and the requirements for the functionality of foreign glycoproteins within viral envelopes, however, have not been elucidated. Conditions for efficient insertion of foreign glycoproteins into the vesicular stomatitis virus (VSV) envelope were first established by inserting the wild-type envelope glycoprotein (G) of VSV expressed by a vaccinia virus recombinant. To determine whether the transmembrane and cytoplasmic portions of the VSV G protein were required for insertion of the HIV receptor, a chimeric CD4/G glycoprotein gene was constructed and a vaccinia virus recombinant which expresses the fused CD4/G gene was isolated. The chimeric CD4/G protein was functional as shown in a syncytium-forming assay in HeLa cells as demonstrated by coexpression with a vaccinia virus recombinant expressing the HIV envelope protein. The CD4/G protein was efficiently inserted into the envelope of VSV, and the virus particles retained their infectivity even after specific immunoprecipitation experiments with monoclonal anti-CD4 antibodies. Expression of the normal CD4 protein also led to insertion of the receptor into the envelope of VSV particles. The efficiency of CD4 insertion was similar to that of CD4/G, with approximately 60 molecules of CD4/G or CD4 per virus particle compared with 1,200 molecules of VSV G protein. Considering that (i) the amount of VSV G protein in the cell extract was fivefold higher than for either CD4 or CD4/G and (ii) VSV G protein is inserted as a trimer (CD4 is a monomer), the insertion of VSV G protein was not significantly preferred over CD4 or CD4/G, if at all. We conclude that the efficiency of CD4 or CD4/G insertion appears dependent on the concentration of the glycoprotein rather than on specific selection of these glycoproteins during viral assembly.     

FDF03, a novel inhibitory receptor of the immunoglobulin superfamily, is expressed by human dendritic and myeloid cells

In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14+/CD1a- DC derived from CD34+ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c+ blood and tonsil DC, but not by CD11c- DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcgammaRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.     

Lack of correlation between soluble CD4-induced shedding of the human immunodeficiency virus type 1 exterior envelope glycoprotein and subsequent membrane fusion events.

The noncovalent association of the gp120 and gp41 envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) is disrupted by soluble CD4 binding, resulting in shedding of the gp120 exterior envelope glycoprotein. This observation has led to the speculation that interaction of gp120 with the CD4 receptor triggers shedding of the exterior envelope glycoprotein, allowing exposure of gp41 domains necessary for membrane fusion steps involved in virus entry or syncytium formation. To test this hypothesis, a set of HIV-1 envelope glycoprotein mutants were used to examine the relationship of soluble CD4-induced shedding of the gp120 glycoprotein to envelope glycoprotein function in syncytium formation and virus entry. All mutants with a threefold or greater reduction in CD4-binding ability exhibited marked decreases in gp120 shedding in response to soluble CD4, even though several of these mutants exhibited significant levels of envelope glycoprotein function. Conversely, most fusion-defective mutants with wild-type gp120-CD4 binding affinity, including those with changes in the V3 loop, efficiently shed gp120 following soluble CD4 binding. Thus, soluble CD4-induced shedding of gp120 is not a generally useful marker for conformational changes in the HIV-1 envelope glycoproteins necessary for the virus entry or syncytium formation processes. Some gp120 mutants, despite being expressed on the cell surface and capable of efficiently binding soluble CD4, exhibited decreased gp120 shedding. These mutants were still sensitive to neutralization by soluble CD4, indicating that, for envelope glycoproteins exhibiting high affinity for soluble CD4, competitive inhibition may be more important than gp120 shedding for the antiviral effect.     

CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression

CD147 is a broadly expressed plasma membrane glycoprotein containing two immunoglobulin-like domains and a single charge-containing transmembrane domain. Here we use co-immunoprecipitation and chemical cross-linking to demonstrate that CD147 specifically interacts with MCT1 and MCT4, two members of the proton-linked monocarboxylate (lactate) transporter family that play a fundamental role in metabolism, but not with MCT2. Studies with a CD2-CD147 chimera implicate the transmembrane and cytoplasmic domains of CD147 in this interaction. In heart cells, CD147 and MCT1 co-localize, concentrating at the t-tubular and intercalated disk regions. In mammalian cell lines, expression is uniform but cross-linking with anti-CD147 antibodies caused MCT1, MCT4 and CD147, but not GLUT1 or MCT2, to redistribute together into 'caps'. In MCT-transfected cells, expressed protein accumulated in a perinuclear compartment, whereas co-transfection with CD147 enabled expression of active MCT1 or MCT4, but not MCT2, in the plasma membrane. We conclude that CD147 facilitates proper expression of MCT1 and MCT4 at the cell surface, where they remain tightly bound to each other. This association may also be important in determining their activity and location.     

Proximity between periplasmic loops in the lactose permease of Escherichia coli as determined by site-directed spin labeling

Site-directed thiol cross-linking indicates that the first periplasmic loop (loop I/II) in the lactose permease of Escherichia coli is in close proximity to loops VII/VIII and XI/XII [Sun, J., and Kaback, H. R. (1997) Biochemistry 36, 11959-11965]. To determine whether thiol cross-linking reflects proximity as opposed to differences in the reactivity and/or dynamics of the Cys residues that undergo cross-linking, single-Cys mutants in loops I/II, VII/VIII, and XI/XII and double-Cys mutants in loop I/II and VII/VIII or XI/XII were purified and labeled with a sulfhydryl-specific nitroxide spin label. The labeled mutants were then analyzed by electron paramagnetic resonance (EPR) spectroscopy, and interspin distance was estimated from the extent of line shape broadening in the double-labeled proteins. Out of six paired double-Cys mutants that exhibit thiol cross-linking, five display significant spin-spin interaction. Furthermore, there is a qualitative correlation between distances estimated by site-directed cross-linking and EPR. Taken as a whole, the results are consistent with the conclusion that site-directed thiol cross-linking is primarily a reflection of proximity.     

Regulation of MHC class II signal transduction by the B cell coreceptors CD19 and CD22

The major histocompatability class II heterodimer (class II) is expressed on the surface of both resting and activated B cells. Although it is clear that class II expression is required for Ag presentation to CD4(+) T cells, substantial evidence suggests that class II serves as a signal transducing receptor that regulates B cell function. In ex vivo B cells primed by Ag receptor (BCR) cross-linking and incubation with IL-4, or B cell lines such as K46-17 micromlambda, class II ligation leads to the activation of protein tyrosine kinases, including Lyn and Syk and subsequent phospholipase Cgamma-dependent mobilization of Ca(2+). In this study, experiments demonstrated reciprocal desensitization of class II and BCR signaling upon cross-linking of either receptor, suggesting that the two receptors transduce signals via common processes and/or effector proteins. Because class II and BCR signal transduction pathways exhibit functional similarities, additional studies were conducted to evaluate whether class II signaling is regulated by BCR coreceptors. Upon cross-linking of class II, the BCR coreceptors CD19 and CD22 were inducibly phosphorylated on tyrosine residues. Phosphorylation of CD22 was associated with increased recruitment and binding of the protein tyrosine phosphatase SHP-1. Similarly, tyrosine phosphorylation of CD19 resulted in recruitment and binding of Vav and phosphatidylinositol 3-kinase. Finally, co-cross-linking studies demonstrated that signaling via class II was either attenuated (CD22/SHP-1) or enhanced (CD19/Vav and phosphatidylinositol 3-kinase), depending on the coreceptor that was brought into close proximity. Collectively, these results suggest that CD19 and CD22 modulate class II signaling in a manner similar to that for the BCR.     

sgp-60, a signal-transducing glycoprotein concerned with T cell activation through the T cell receptor/CD3 complex

T cell activation depends not only on the expression of a TCR, but also on that of accessory molecules that function in cell-cell adhesion and/or signal transduction. The subject of this report is the biochemical and functional characterization of what appears to be a novel murine lymphocyte cell surface antigen, provisionally termed sgp-60. Extensive, higher-order cross-linking of this glycoprotein with an anti-sgp-60 mAb and a second-step antibody reagent results in the activation of resting CD4+ T cells in the presence of a second signal. Monovalent or bivalent engagement of sgp-60 by the anti-sgp-60 antibody results in profound and direct inhibition of anti-CD3- or Con A-driven T cell activation, whereas alternative T cell activation via the phosphatidylinositol-linked proteins Thy-1 and TAP/Ly-6A is not affected. These findings raise the possibility that the sgp-60 molecule may be specifically involved in signal transduction through the TCR/CD3 complex and thus point to an important physiologic role for this protein in CD4+ T cells.     

Characterization of the noncovalent complex of human immunodeficiency virus glycoprotein 120 with its cellular receptor CD4 by matrix-assisted laser desorption/ionization mass spectrometry.

The initial event in infection by the human immunodeficiency virus type 1 (HIV-1) is the interaction of the viral envelope glycoprotein (HIV-gp120) with its primary cellular receptor, the glycoprotein CD4. Molecular structure information about the HIV-gp120/CD4 complex can provide information relevant to an understanding of the basic processes occurring in HIV infection and to development of therapies that can inhibit AIDS. Previous studies by sugar gradient sedimentation of the interaction of HIV-gp120 with a cytoplasmic domain truncated soluble CD4 (sCD4) suggested that a one-to-one complex was formed. The stoichiometry, however, of the sCD4/HIV-gp120 complex remained to be confirmed by an independent method because (i) recent X-ray examination revealed dimerization of sCD4 and (ii) the low resolution and low accuracy of molecular weight determination by sugar gradient sedimentation can lead to artifactual data. Therefore, in this study matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to determine the molecular mass of the complex of fully glycosylated HIV-gp120 and sCD4. A mass of 145 kDa was measured, which is exactly the sum of the molecular masses of one HIV-gp120 and one sCD4 molecule. Complexes of higher order of stoichiometry were not detected. Identical results were obtained by chemically cross-linking the HIV-gp120/sCD4 complex with subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and MALDI-MS. This study confirms the earlier suggestions of the stoichiometry of the sCD4/HIV-gp120 complex in solution and also demonstrates the potential of MALDI-MS in investigations of specific noncovalent complexes of glycoproteins.     

Subunit composition of the membrane glycoprotein complex of Semliki Forest virus

The quaternary structure of the membrane glycoproteins E1, E2 and E3 of Semliki Forest virus has been determined in intact virus and in the protein complexes obtained after Triton X100 solubilization. Intact and solubilized virus were treated with a cleavable cross-linking reagent and the covalently cross-linked glycoprotein complexes were isolated and characterized using antibodies specific for the E1 and E2 membrane glycoproteins. The isolation and characterization procedure was done in a low sodium dodecyl sulphate concentration which prevented non-covalent association between glycoprotein species, but did not abolish antigen-antibody binding.The major glycoprotein complex seen after cross-linking of either intact or Triton X100 solubilized virus was an approximately 100,000 molecular weight species composed of E1-E2 heterodimers only. These findings show that E1 and E2 form a complex in the virus and that this complex is retained after solubilization with Triton X100. The smallest membrane glycoprotein E3 was not cross-linked to the other proteins and was therefore lost in the isolation procedure. However, the presence of E3 together with E1 and E2 in complexes obtained after Triton X100 solubilization of intact virus suggests that an E1-E2-E3 trimer is present in the virus. It is likely that this trimer forms the spike-like structures seen on the surface of the virus.We have observed that antibody specific for one component of the virus glycoprotein complex can induce rearrangement of uncross-linked complexes in Triton X100 solubilized form. This fact should be considered when using specific antibody for characterization of protein complexes.     

Contribution of the N-linked carbohydrate of erythrocyte antigen CD59 to its complement-inhibitory activity.

The contribution of N-linked carbohydrate to the complement-inhibitory function of the human erythrocyte membrane glycoprotein, CD59, was investigated. Amino acid sequence analysis of tryptic peptides labeled with [3H]borohydride revealed an N-linked carbohydrate moiety at the Asn18 residue. No O-linked carbohydrate was detected, as judged by the failure of asialo-CD59 to bind peanut agglutinin and by its resistance to digestion by O-glycanase. The apparent molecular mass of CD59 was reduced from 18-20 to 14 kDa upon complete digestion with N-glycanase, with no detectable proteolysis. N-glycanase digestion of CD59 was associated with an 88 +/- 4% loss of the complement-inhibitory activity of the protein, as assessed by its capacity to protect chicken erythrocytes from lysis by the human C5b-9 proteins. By contrast, no change in function was observed after digestion of CD59 with neuraminidase, under conditions that removed greater than 60% of [3H]sialic acid residues. Despite loss of functional activity after N-glycanase digestion, we detected no change in the capacity of the deglycosylated CD59 to incorporate into erythrocyte membranes or to bind specifically and with species selectivity to the C8 and C9 components of the membrane attack complex. In order to alter the branched-chain structure of the N-linked carbohydrate of CD59 without enzymatic digestion, Chinese hamster ovary (CHO) cells transfected with cDNA for human CD59 were grown in the alpha-mannosidase inhibitor, 1-deoxymannojirimycin, resulting in conversion of approximately 70% of the membrane glycoprotein to a high mannose. When grown in the presence of 1-deoxymannojirimycin, the C5b-9-inhibitory activity of CD59 expressed on the surface of the transfected CHO cells was reduced by an amount comparable to that observed for the N-glycanase digested protein. Taken together, these data suggest that normal glycosylation of Asn18 in CD59 is required for the normal expression of its complement-inhibitory activity on membrane surfaces, although these N-linked sugar residues do not contribute to CD59's affinity for the C8 and C9 components of the C5b-9 complex.     

Synthesis of the glycosylated polypeptide chain of an inducible costimulator on T-cells

The glycoprotein AILIM/ICOS (Activation inducible lymphocyte immunomediately molecule/Inducible co-stimulator) on T-cells was identified in 1998 as a member of the CD28/CTLA4 family. The three-dimensional structure of the AILIM/ICOS extracellular domain has not been solved, and therefore we have examined the preparation of homogeneous glycosylated polypeptide chains of this domain having two homogeneous N-linked complex type oligosaccharides for use in folding experiments. To synthesize the glycosylated whole polypeptide chain of the AILIM/ICOS extracellular domain, the target polypeptide chain was divided into four segments, each containing a cysteine residue. Those peptide segments were synthesized by conventional SPPS, followed by thioesterification of the C-terminus. The oligosaccharide moiety, a biantennary complex type disialyloligosaccharide, was attached to the cysteine thiol in the peptide backbone using the haloacetamide method. These peptides, as well as a glycosylated peptide, were sequentially coupled by use of native chemical ligation. This process successfully afforded the desired polypeptide chain having homogeneous oligosaccharides.