Complex-type Sugars Research Articles

Tobacco seeds as efficient production platform for a biologically active anti-HBsAg monoclonal antibody.

The use of plants as heterologous hosts is one of the most promising technologies for manufacturing valuable recombinant proteins. Plant seeds, in particular, constitute ideal production platforms for long-term applications requiring a steady supply of starting material, as they combine the general advantages of plants as bioreactors with the possibility of biomass storage for long periods in a relatively small volume, thus allowing manufacturers to decouple upstream and downstream processing. In the present work we have used transgenic tobacco seeds to produce large amounts of a functionally active mouse monoclonal antibody against the Hepatitis B Virus surface antigen, fused to a KDEL endoplasmic reticulum retrieval motif, under control of regulatory sequences from common bean (Phaseolus vulgaris) seed storage proteins. The antibody accumulated to levels of 6.5mg/g of seed in the T3 generation, and was purified by Protein A affinity chromatography combined with SEC-HPLC. N-glycan analysis indicated that, despite the KDEL signal, the seed-derived plantibody bore both high-mannose and complex-type sugars that indicate partial passage through the Golgi compartment, although its performance in the immunoaffinity purification of HBsAg was unaffected. An analysis discussing the industrial feasibility of replacing the currently used tobacco leaf-derived plantibody with this seed-derived variant is also presented.

Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope.

The gp120/gp41 HIV-1 envelope glycoprotein (Env) is highly glycosylated, with up to 50% of its mass consisting of N-linked glycans. This dense carbohydrate coat has emerged as a promising vaccine target, with its glycans acting as epitopes for a number of potent and broadly neutralizing antibodies (bnAbs). Characterizing the glycan structures present on native HIV-1 Env is thus a critical goal for the design of Env immunogens. In this study, we used a complementary, multistep approach involving ion mobility mass spectrometry and high-performance liquid chromatography to comprehensively characterize the glycan structures present on HIV-1 gp120 produced in peripheral blood mononuclear cells (PBMCs). The capacity of different expression systems, including pseudoviral particles and recombinant cell surface trimers, to reproduce native-like glycosylation was then assessed. A population of oligomannose glycans on gp120 was reproduced across all expression systems, supporting this as an intrinsic property of Env that can be targeted for vaccine design. In contrast, Env produced in HEK 293T cells failed to accurately reproduce the highly processed complex-type glycan structures observed on PBMC-derived gp120, and in particular the precise linkage of sialic acid residues that cap these glycans. Finally, we show that unlike for gp120, the glycans decorating gp41 are mostly complex-type sugars, consistent with the glycan specificity of bnAbs that target this region. These findings provide insights into the glycosylation of native and recombinant HIV-1 Env and can be used to inform strategies for immunogen design and preparation. Development of an HIV vaccine is desperately needed to control new infections, and elicitation of HIV bnAbs will likely be an important component of an effective vaccine. Increasingly, HIV bnAbs are being identified that bind to the N-linked glycans coating the HIV envelope glycoproteins gp120 and gp41, highlighting them as important targets for vaccine design. It is therefore important to characterize the glycan structures present on native, virion-associated gp120 and gp41 for development of vaccines that accurately mimic native-Env glycosylation. In this study, we used a number of analytical techniques to precisely study the structures of both the oligomannose and complex-type glycans present on native Env to provide a reference for determining the ability of potential HIV immunogens to accurately replicate the glycosylation pattern on these native structures.

P144 Pathogen recognition by the long pentraxin PTX3: Roles of glycosylation and interplay with complement

Introduction The long pentraxin PTX3 is a soluble Pattern Recognition Molecule (sPRM) with non-redundant roles in resistance to selected pathogens, such as the opportunistic fungus Aspergillus fumigatus . PTX3 facilitates recognition and phagocytosis of fungal conidia mainly by polymorphonuclear neutrophils (PMNs) via Fc γ receptors (Fc γ Rs) and alternative complement pathway (ACP) mechanisms. The human PTX3 protein contains a single N-glycosylation site with complex type sialylated oligosaccharides that has been involved in a number of PTX3 functions [1] . The present study was designed to characterize the role of this post translational modification in the PTX3-depedent recognition and phagocytosis of A. fumigatus . This microorganism has developed several strategies to evade the immune response, such as attracting onto the conidial wall inhibitors of the ACP, like factor H (FH, a ligand of PTX3). Here we present preliminary evidences on the effect of PTX3 on FH engagement by A. fumigatus . Methods Recombinant as well as natural human PTX3 (i.e., from PMNs) was purified by immunoaffinity chromatography and the attached oligosaccharides were analyzed using glycosidases, lectin blotting and mass spectrometry. A range of PTX3 glycoforms were made in vitro by enzymatic hydrolysis of terminal monosaccharides of the recombinant protein. The opsonic and pro-phagocytic activities of these variants were characterized in buffer solutions and whole human peripheral blood, respectively, by flow cytometry. The influence of PTX3 on FH binding to conidia was studied in competition assays using microtiter plates. Results Complex-type sugars were found on PTX3 of both natural and recombinant origins. These showed heterogeneity with regard to the relative content of terminal sialic acid (SA) and bi-, tri- and tetrantennary glycans, depending on the cellular source. Enzymatic removal of SA caused ∼50% reduction of PTX3 binding to A. fumigatus conidia, with no further change due to additional deglycosylation. Conversely, saccharide hydrolysis had no effect on PTX3 binding to PMNs, where Fc γ Rs have been proposed as putative receptors of this pentraxin [1] . However, consistent with binding data from conidia, desialylated PTX3 showed reduced potentiation of conidia phagocytosis by PMNs as compared to the unmodified protein. Also, we observed that PTX3 inhibits the binding of FH to A. fumigatus conidia, even when these were pre-opsonised with the long pentraxin and FH was applied at physiologically relevant concentrations. Conclusion Different pathogens express neuraminidases on their surface during infection and natural immunity cells, such as PMNs, mobilize intracellular neuraminidases to the plasma membrane upon inflammation. Here, we report that the glycosylation status of the sPRM PTX3 changes, with respect to SA, depending on cellular source and perhaps inducing stimuli. We describe that SA modulates the PTX3-dependent recognition and phagocytosis of A. fumigatus , which points to glycosylation as a strategy to tune PTX3 functions in innate immunity. Furthermore, we provided evidences that PTX3 impairs the interaction of FH with A. fumigatus , where this might link to a new mechanism to counterbalance the complement evasion strategy exploited by this pathogen.

Production of different glycosylation variants of the tumour-targeting mAb H10 in Nicotiana benthamiana: influence on expression yield and antibody degradation

We previously described the expression of a tumour-targeting antibody (mAb H10) in Nicotiana benthamiana by vacuum-agro-infiltration and the remarkable yields of highly pure protein achieved. The objective of the present work was to investigate different strategies for transient overexpression of the mAb H10 in which glycan configuration was modulated and assess how these strategies affect the accumulation yield and stability of the antibody. To this aim, three procedures have been assayed: (1) Site-directed mutagenesis to abolish the glycosylation site; (2) endoplasmic reticulum retention (C-terminal SEKDEL fusion) to ensure predominantly high-mannose type glycans; and (3) expression in a N. benthamiana RNAi down-regulated line in which β1,2-xylosyltransferase and α1,3-fucosyltransferase gene expression is silenced. The three antibody variants (H10-Mut) (H10-SEKDEL) (H10(XylT/FucT)) were transiently expressed, purified and characterised for their glycosylation profile, expression/purification yield and antibody degradation pattern. Glycosylation analysis of H10(XylT/FucT) demonstrated the absence of plant complex-type sugars, while H10-SEKDEL, although substantially retained in the ER, revealed the presence of β1,2-xylose and α1,3-fucose residues, indicating a partial escape from the ER retrieval system. Antibody accumulation and purification yields were not enhanced by ER retention. All H10 antibody glyco-forms revealed greater degradation compared to the original, resulting mostly in the formation of Fab fragments. In the case of aglycosylated H10-Mut, more than 95% of the heavy chain was cleaved, confirming the pivotal role of the sugar moiety in protein stability. Identification of possible 'fragile' sites in the H10 antibody hinge region could be of general interest for the development of new strategies to reduce antibody degradation and increase the yield of intact IgGs in plants.

New insights into the functions and N‐glycan structures of factor X activator from Russell’s viper venom

The coagulation factor X activator from Russell's viper venom (RVV-X) is a heterotrimeric glycoprotein. In this study, its three subunits were cloned and sequenced from the venom gland cDNAs of Daboia siamensis. The deduced heavy chain sequence contained a C-terminal extension with four additional residues to that published previously. Both light chains showed 77-81% identity to those of a homologous factor X activator from Vipera lebetina venom. Far-western analyses revealed that RVV-X could strongly bind protein S, in addition to factors X and IX. This might inactivate protein S and potentiate the disseminated intravascular coagulation syndrome elicited by Russell's viper envenomation. The N-glycans released from each subunit were profiled and sequenced by MALDI-MS and MS/MS analyses of the permethyl derivatives. All the glycans, one on each light chain and four on the heavy chain, showed a heterogeneous pattern, with a combination of variable terminal fucosylation and sialylation on multiantennary complex-type sugars. Amongst the notable features were the presence of terminal Lewis and sialyl-Lewis epitopes, as confirmed by western blotting analyses. As these glyco-epitopes have specific receptors in the vascular system, they possibly contribute to the rapid homing of RVV-X to the vascular system, as supported by the observation that slower and fewer fibrinogen degradation products are released by desialylated RVV-X than by native RVV-X.

Human Follicular Lymphoma Cells Contain Oligomannose Glycans in the Antigen-binding Site of the B-cell Receptor

Expression of surface immunoglobulin appears critical for the growth and survival of B-cell lymphomas. In follicular lymphoma, we found previously that the Ig variable (V) regions in the B-cell receptor express a strikingly high incidence of N-glycosylation sequons, NX(S/T). These potential glycosylation sites are introduced by somatic mutation and are lymphoma-specific, pointing to their involvement in tumor pathogenesis. Analysis of the V region sugars from lymphoma-derived IgG/IgM reveals that they are mostly oligomannose and, remarkably, are located in the antigen-binding site, possibly precluding conventional antigen binding. The Fc region contains complex glycans, confirming that the normal glycan processing pathway is intact. Binding studies indicate that the oligomannose glycans occupying the V regions are accessible to mannose-binding lectin. These findings suggest a potential contribution to lymphoma pathogenesis involving antigen-independent interaction of surface immunoglobulin of the B-cell receptor with mannose-binding molecules of innate immunity in the germinal center.

Structure and Function of the Long Pentraxin PTX3 Glycosidic Moiety: Fine-Tuning of the Interaction with C1q and Complement Activation

The prototypic long pentraxin PTX3 is a unique fluid-phase pattern recognition receptor that plays a nonredundant role in innate immunity and female fertility. The PTX3 C-terminal domain is required for C1q recognition and complement activation and contains a single N-glycosylation site on Asn 220. In the present study, we characterized the structure of the human PTX3 glycosidic moiety and investigated its relevance in C1q interaction and activation of the complement classical pathway. By specific endo and exoglycosidases digestion and direct mass spectrometric analysis, we found that both recombinant and naturally occurring PTX3 were N-linked to fucosylated and sialylated complex-type sugars. Interestingly, glycans showed heterogeneity mainly in the relative amount of bi, tri, and tetrantennary structures depending on the cell type and inflammatory stimulus. Enzymatic removal of sialic acid or the entire glycosidic moiety equally enhanced PTX3 binding to C1q compared to that in the native protein, thus indicating that glycosylation substantially contributes to modulate PTX3/C1q interaction and that sialic acid is the main determinant of this contribution. BIAcore kinetic measurements returned decreasing K(off) values as sugars were removed, pointing to a stabilization of the PTX3/C1q complex. No major rearrangement of PTX3 quaternary structure was observed after desialylation or deglycosylation as established by size exclusion chromatography. Consistent with C1q binding, PTX3 desialylation enhanced the activation of the classical complement pathway, as assessed by C4 and C3 deposition. In conclusion, our results provided evidence of an involvement of the PTX3 sugar moiety in C1q recognition and complement activation.

Sugar Binding Properties of the Two Lectin Domains of the Tandem Repeat-type Galectin LEC-1 (N32) of Caenorhabditis elegans: DETAILED ANALYSIS BY AN IMPROVED FRONTAL AFFINITY CHROMATOGRAPHY METHOD

The 32-kDa galectin (LEC-1 or N32) of the nematode Caenorhabditis elegans is the first example of a tandem repeat-type galectin and is composed of two domains, each of which is homologous to typical vertebrate 14-kDa-type galectins. To elucidate the biological meaning of this unique structure containing two probable sugar binding sites in one molecule, we analyzed in detail the sugar binding properties of the two domains by using a newly improved frontal affinity chromatography system. The whole molecule (LEC-1), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) were expressed in Escherichia coli, purified, and immobilized on HiTrap gel agarose columns, and the extent of retardation of various sugars by the columns was measured. To raise the sensitivity of the system, we used 35 different fluorescence-labeled oligosaccharides (pyridylaminated (PA) sugars). All immobilized proteins showed affinity for N-acetyllactosamine-containing N-linked complex-type sugar chains, and the binding was stronger for more branched sugars. Ch showed 2-5-fold stronger binding toward all complex-type sugars compared with Nh. Both Nh and Ch preferred Galbeta1-3GlcNAc to Galbeta1-4GlcNAc. Because the Fucalpha1-2Galbeta1-3GlcNAc (H antigen) structure was found to interact with all immobilized protein columns significantly, the K(d) value of pentasaccharide Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-PA for each column was determined by analyzing the concentration dependence. Obtained values for immobilized LEC-1, Nh, and Ch were 6.0 x 10(-5), 1.3 x 10(-4), and 6.5 x 10(-5) m, respectively. The most significant difference between Nh and Ch was in their affinity for GalNAcalpha1-3(Fucalpha1-2)Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-PA, which contains the blood group A antigen; the K(d) value for immobilized Nh was 4.8 x 10(-5) m, and that for Ch was 8.1 x 10(-4) m. The present results clearly indicate that the two sugar binding sites of LEC-1 have different sugar binding properties.

Male-specific Modification of Human CD52

CD52 is an unusually short, bipolar glycopeptide bearing a highly charged N-linked carbohydrate moiety and a glycosylphosphatidylinositol membrane anchor. It is exclusively expressed on lymphocytes and in the male genital tract where it is shed into the seminal plasma and inserts into the sperm membrane. The sperm surface molecule has potential significance as a target for antibodies that inhibit sperm function and gamete interaction. Western blot analyses suggested cell type-specific modifications of the antigen. It was purified from seminal plasma and a detailed structural analysis performed. The majority of anchor structures in male genital tract CD52 showed 2-inositol palmitoylation, rendering molecules insensitive toward phospholipase C, and a sn-1-alkyl-2-lyso-glycerol structure in place of the diacylated anchor described by Treumann et al. (Treumann, A., Lifely, M. R., Schneider, P., and Ferguson, M. A. (1995) J. Biol. Chem. 270, 6088-6099). N-Glycans of the male genital tract product were based on bi-, tri-, and tetraantennary structures of highly charged (up to -7), terminally sialylated complex-type sugars. A substantial proportion carried varying numbers of lactosamine repeats of which nearly 30% were branched. Different from lymphocytes, 10-15% of all N-glycans of the male genital tract antigen also contained peripheral fucose. These data confirm that male genital tract CD52 is distinct from the lymphocyte form by both N-linked glycans and COOH-terminal attached lipid anchor.

Analysis of carbohydrate residues on recombinant human thyrotropin receptor.

An investigation of the sugar groups on recombinant human TSH receptors (TSHR) expressed in CHO-K1 cells and solubilized with detergents is described. Western blotting studies with TSHR monoclonal antibodies showed that the receptor was present principally as two bands with approximate molecular masses of 120 and 100 kDa. Further blotting studies using lectins and/or involving treatment with different glycosidases indicated that the 100-kDa band contained about 16 kDa of high mannose-type sugars, and the 120-kDa band contained about 33 kDa of complex-type sugars. It was possible to separate the 120- and 100-kDa components of the TSHRs by lectin affinity chromatography. In particular, Galanthus nivalis lectin, which binds high mannose-type sugars, bound the 100-kDa band, but not the 120-kDa band, whereas Datura stramonium lectin, which binds complex-type sugars, bound the 120-kDa band, but not the 100-kDa band. 125I-Labeled TSH binding studies with the various lectin column fractions showed that TSH-binding activity was principally associated with the complex-type sugar containing the 120-kDa form of the receptor rather than the high mannose-containing 100-kDa form. During peptide chain glycosylation, high mannose-type sugar residues are attached first and then modified by the formation of complex type structures to form the mature glycoprotein. Our data suggest that in the case of the TSH receptor, this type of posttranslational processing has an important role in forming the TSH-binding site.

Effect of glycosylation on the stability of α1-antitrypsin toward urea denaturation and thermal deactivation

The effects of glycosylation on the stability of human α 1-antitrypsin were investigated. The transition midpoints in urea-induced equilibrium unfolding of a non-glycosylated recombinant, a yeast version of glycosylated, and human plasma α 1-antitrypsin were 1.8 M, 2.2 M, and 2.5 M at 25°C, respectively. Kinetic analyses of unfolding and refolding revealed that glycosylation retarded the unfolding without affecting the refolding rate significantly, suggesting that the stability increase is due to the stabilization of the native state as opposed to the destabilization of the unfolded state. In thermal deactivation, which is a heat-induced aggregation process, the ungycosylated recombinant α 1-antitrypsin was deactivated most easily, which was followed in order by the yeast, and the plasma form. The results indicate that glycosylation confers the increase in stability of α 1-antitrypsin, and that the oligomannose sugars present on the yeast form produce a less stable molecule than the complex type sugars on the plasma form. It appears that the effect of glycosylation on the enhancement of thermal resistance is exerted through the increase in conformational stability. However, a stable recombinant variant (Phe 51→Cys) that showed the same conformational stability as the plasma form was less resistant to thermal denaturation than the plasma α 1-antitrypsin. The results suggest that the existence of carbohydrate moiety per se as well as the conformational stability contribute to the kinetic stability of α 1-antitrypsin toward aggregation.

Biosynthesis and intracellular transport of a membrane glycoprotein (plgp57) of the prelysosome compartment.

We have characterized the biosynthesis and intracellular transport of a membrane glycoprotein, designated plgp57, which is found predominantly in the prelysosome compartment (PLC) of Madin-Darby bovine kidney cells. In pulse-chase experiments, plgp57 was found to be initially synthesized as a 35 kDa precursor which was modified to yield a diffuse approximately 57 kDa mature form. Digestion with endoglycosidase H (endo H) demonstrated that the 35 kDa precursor contained three endo H-sensitive, high mannose-type oligosaccharides which became modified to endo H-resistant, complex-type sugars on the approximately 57 kDa mature form. Labelling cells in the presence of tunicamycin and treatment of the 35 kDa precursor with endo H revealed that plgp57 has a core protein of 24 kDa to which three N-asparagine-liked oligosaccharides are attached. Other experiments indicated that plgp57 could be differentially glycosylated on a common 24 kDa core protein in different cell types. The half-lives of the glycosylated and non-glycosylated forms of plgp57 were approximately 18 and approximately 13 h, respectively. Glycosylated and non-glycosylated plgp57 exhibited similar steady-state intracellular distributions, indicating that targeting of plgp57 to the PLC does not require carbohydrate address markers. Pulse-labeling of cells followed by organelle fractionation at various chase times revealed a t1/2 = approximately 1 h for the transit of newly synthesized plgp57 to the PLC. Finally, amino terminal sequencing of plgp57 revealed the similarity of this protein to the CD63/ME491 family of membrane glycoproteins.

Intracellular processing of the human respiratory syncytial virus fusion glycoprotein: amino acid substitutions affecting folding, transport and cleavage.

The intracellular processing and transport of the respiratory syncytial virus (RSV) fusion (F) glycoprotein was examined by comparing the maturation and stability of wild-type F, uncleaved mutant F and chimeric F glycoproteins expressed by recombinant vaccinia viruses to that of F protein expressed by RSV. One of the recombinant viruses, vF317, expressed F protein (F317) that was processed like the RSV F glycoprotein. F317 was synthesized initially as F0, the uncleaved glycosylated precursor of mature F protein, and formed stable oligomeric structures that were maintained following cleavage of F0 to form the disulphide bond-linked F1 and F2 subunits. Most of the newly synthesized F0 expressed by either RSV or by vF317 was sensitive to treatment with endoglycosidase H (Endo H). Following cleavage of F0, F1 was resistant to Endo H, suggesting that conversion to complex-type sugars, which takes place in the medial Golgi apparatus, occurred simultaneously with or immediately prior to cleavage of F0 into F1 and F2. Another recombinant virus, vF313, synthesized only uncleaved F protein (F313) that comigrated with F0. Uncleaved F313 was expressed as a stable glycosylated protein; however, unlike cleaved F317, its oligosaccharides were not modified to complex forms, as determined from its Endo H sensitivity, and uncleaved F313 did not assemble into stable oligomeric structures. Nucleotide sequence analysis of the cDNA clones encoding F313 and F317 revealed four predicted amino acid sequence differences, none of which were located at the cleavage site. Expression of chimeric F proteins obtained by restriction fragment exchange between the two cDNA clones indicated that two amino acid changes in the F1 domain, located at amino acid residues 301 (Val to Ala) and 447 (Val to Met), resulted in the expression of uncleaved F protein. A change at either of these two amino acid residues, 301 or 447, resulted in the expression of inefficiently cleaved F protein, defining an additional F protein phenotype. Pulse-chase analyses to examine the association of recombinant F glycoproteins with gradient-purified fractionated membranes or with GRP78-BiP, a protein resident in the endoplasmic reticulum (ER) which binds to nascent proteins, revealed that uncleaved F protein (F313) is associated with GRP78-BiP in the ER for a longer time than F317, and little if any F313 was transported to the cell surface. In addition, the uncleaved F protein (F313) was not recognized by a panel of F protein-specific monoclonal antibodies in ELISA or indirect immunofluorescence assays, suggesting that F313 was misfolded and, as a result, not transported properly or cleaved.

Friend spleen focus-forming virus glycoprotein gp55 interacts with the erythropoietin receptor in the endoplasmic reticulum and affects receptor metabolism.

The Friend spleen focus-forming virus envelope glycoprotein, gp55, binds to the murine erythropoietin receptor (EPO-R) and triggers growth activation in the absence of EPO. Interleukin 3-dependent lymphoid cell lines that have been stably transfected with the EPO-R cDNA grow in the presence of EPO or interleukin 3. In these cells, the EPO-R is synthesized as a minor 62-kDa unglycosylated form and a major 64-kDa form carrying one high-mannose N-linked oligosaccharide. A fraction of the 64-kDa form is processed to a 66-kDa species with complex-type sugars. Very little of the EPO-R is expressed on the cell surface and all three forms of EPO-R are degraded rapidly. Cells transfected with both EPO-R and gp55 cDNAs grow in the absence of EPO. Most of the EPO-R associated with gp55 is endoglycosidase H-sensitive, suggesting that the interactions between these proteins occur in the endoplasmic reticulum. Furthermore, the endoglycosidase H-sensitive EPO-R is more stable than in the absence of gp55, a result suggesting that interaction of gp55 with the EPO-R causes it to remain within the rough endoplasmic reticulum. It is possible that gp55 EPO-R complexes within this compartment send a growth-promoting signal to the cell.

Recycling glycoproteins do not return to the cis-Golgi.

Recycling of a number of glycoproteins along the site of action of mannosidase I (the distal endoplasmic reticulum/cis-Golgi) was followed in several different cell lines. Treatment of cells with 1-deoxymannojirimycin (dMM) produced high mannose oligosaccharides at positions otherwise occupied by complex-type oligosaccharides in these glycoproteins. Conversion of high-mannose-type oligosaccharides to complex oligosaccharides of proteins initially synthesized in the presence of dMM was used as a marker for recycling of glycoproteins along the site of action of dMM. In contrast to findings reported by Snider and Rogers (Snider, M. D., and O. C. Rogers. 1986. J. Cell Biol. 103:265-275), removal of dMM did not result in reconversion of high-mannose oligosaccharides to complex-type sugars, even after prolonged periods of culture. We conclude that surface glycoproteins do not recycle through the cis-medial Golgi elements.

Response of aggregating chick corneal cells to modifiers of N-linked oligosaccharides, endoglycosidase H and deoxymannojirimycin.

Chick corneal epithelium takes on its mature conformation between 11 and 16 days of incubation. Earlier work has shown that desmosome frequency increases during this period, reaching its highest rate at 15 1/2 days. In the present report aggregation rates of cells from embryos of 11 days and those of 15 1/2 days are compared. Younger cells, which form fewer desmosomes, aggregate at a more moderate rate than older cells. In addition, younger cells bind less concanavalin A (ConA) than older cells. To determine if increase in ConA binding could be related to these cellular responses, aggregating cells were exposed to endoglycosidase H (EndoH) and to deoxymannojirimycin. This treatment should permit comparison of the response of cells that have a normal complement of N-linked oligosaccharides with those that have reduced high-mannose or complex type sugars. The effectiveness of EndoH under the conditions used was confirmed by failure of treated glycoprotein after separation by SDS-PAGE and electroblotting to bind ConA. Aggregation rates of both older and younger cells were unaffected, as measured by disapperance of single cells, though older cells formed somewhat smaller aggregates at the highest dosage used. Desmosome formation was markedly reduced in the presence of the enzyme, even in the absence of other changes in the fine structure. At the highest dose of the enzyme the fine structure of older but not younger cells showed indications of blockage of transport. Deoxymannojirimycin appears to cause a build-up of high-mannose groups, since treated cells showed increased incorporation of [3H]mannose.(ABSTRACT TRUNCATED AT 250 WORDS)