N-glycosylation affects the adhesive function of E-Cadherin through modifying the composition of adherens junctions (AJs) in human breast carcinoma cell line MDA-MB-435

E-cadherin mediates calcium-dependent cell-cell adhesion between epithelial cells. The ectodomain of human E-cadherin contains four potential N-glycosylation sites at Asn residues 554, 566, 618, and 633. In this study, the role of N-glycosylation in E-cadherin-mediated cell-cell adhesion was investigated by site-directed mutagenesis. In MDA-MB-435 cells, all four potential N-glycosylation sites of human E-cadherin were N-glycosylated. Removal of N-glycan at Asn-633 dramatically affected E-cadherin stability. In contrast, mutant E-cadherin lacking the other three N-glycans showed similar protein stability in comparison with wild-type E-cadherin. Moreover, N-glycans at Asn-554 and Asn-566 were found to affect E-cadherin-mediated calcium-dependent cell-cell adhesion, and removal of either of the two N-glycans caused a significant decrease in calcium-dependent cell-cell adhesion accompanied with elevated cell migration. Analysis of the composition of adherens junctions (AJs) revealed that removal of N-glycans on E-cadherin resulted in elevated tyrosine phosphorylation level of beta-catenin and reduced beta- and alpha-catenins at AJs. These findings demonstrate that N-glycosylation may affect the adhesive function of E-cadherin through modifying the composition of AJs.     

Structural requirements of a human deoxyribonuclease II for the development of the active enzyme form, revealed by site-directed mutagenesis

Using site-directed mutagenesis, we eliminated three potential N-glycosylation sites (N86, N212, and N266) of human deoxyribonuclease II (DNase II), conserved in mammalian enzymes, and a proteolytic processing site (Q46-R47), forming a propeptide subunit of the enzyme. We expressed a series of these mutant DNase II constructs in COS-7 and Hep G2 cells. Liberation of each glycosylation site at N86 and N266 and the cleavage site interfered dramatically with expression of the intracellular and secreted DNase II activities, irrespective of cell line transfected. A chimeric mutant in which the signal peptide of the DNase II was replaced with that of human DNase I had no intracellular or secreted enzyme activity. Therefore, a simultaneous attachment of a carbohydrate moiety to N86 and N266, cleavage of the propeptide from the single DNase II precursor, and the inherent signal peptide might be required for subcellular sorting and proteolytic maturation of the enzyme.     

Vacuolar processing enzyme is self-catalytically activated by sequential removal of the C-terminal and N-terminal propeptides

A vacuolar processing enzyme (VPE) responsible for maturation of various vacuolar proteins is synthesized as an inactive precursor. To clarify how to convert the VPE precursor into the active enzyme, we expressed point mutated VPE precursors of castor bean in the pep4 strain of Saccharomyces cerevisiae. A VPE with a substitution of the active site Cys with Gly showed no ability to convert itself into the mature form, although a wild VPE had the ability. The mutated VPE was converted by the action of the VPE that had been purified from castor bean. Substitution of the conserved Asp-Asp at the putative cleavage site of the C-terminal propeptide with Gly-Gly abolished both the conversion into the mature form and the activation of the mutated VPE. In vitro assay with synthetic peptides demonstrated that a VPE exhibited activity towards Asp residues and that a VPE cleaved an Asp-Gln bond to remove the N-terminal propeptide. Taken together, the results indicate that the VPE is self-catalytically maturated to be converted into the active enzyme by removal of the C-terminal propeptide and subsequent removal of the N-terminal one.     

Functional analysis of the C-terminal propeptide of keratinase from Bacillus licheniformis BBE11-1 and its effect on the production of keratinase in Bacillus subtilis

The keratinase from Bacillus licheniformis BBE11-1 is a serine protease and expressed as a pre-pro-precursor. To produce a mature and active keratinase, the propeptide must be cleaved on the C-terminal via cis or trans. In this study, to enhance the production of keratinase in Bacillus subtilis, single amino acid substitutions, single residue deletions and linkers were introduced at the C-terminus of the propeptide. The results showed that optimizing the residue of cleavage site of propeptide will affect the cleavage efficiency of propeptide, and the mature enzyme yield of Leu(P1)Ala mutant increases 50% compared with the wild-type. In addition, inserting linkers and deleting individual residues at the C-terminal of the propeptide decreases the mature keratinase production. Our results indicated that the primary structure of the C-terminus of propeptide is crucial for the mature keratinase production. Propeptide engineering at C-terminus may be an effective approach to increase the yield of keratinase.     

Asparagine-linked oligosaccharide-independent secretion of egg envelope glycoprotein ZPC of the Japanese quail (Coturnix japonica)

In avian species, a glycoprotein homologous to mammalian ZPC is synthesized in the granulosa cells of developing follicles. We have previously reported that the newly synthesized ZPC (proZPC) in the granulosa cells is cleaved at the consensus furin cleavage site to generate mature ZPC prior to secretion. In the present study, we examined the role of asparagine (N)-linked oligosaccharides in the proteolytic processing of proZPC and the subsequent secretion of ZPC by using site-directed mutagenesis of the consensus sequence for N-glycosylation, and tunicamycin, an inhibitor for N-glycosylation of glycoprotein. Western blot analysis demonstrated that tunicamycin did not block either proteolytic cleavage of proZPC or the subsequent ZPC secretion. Moreover, a site-directed mutant that possesses a mutated sequence for N-glycosylation was efficiently secreted from the cells. These results indicate that proteolytic cleavage of proZPC, and the subsequent ZPC secretion occur in the absence of N-linked oligosaccharides. Therefore, the addition of N-glycans to ZPC polypeptide is not required for quail ZPC secretion.     

N-glycosylation at Asn residues 554 and 566 of E-cadherin affects cell cycle progression through extracellular signal-regulated protein kinase signaling pathway

E-cadherin, which has a widely acknowledged role in mediating calcium-dependent cell-cell adhesion between epithelial cells, also functions as a tumor suppressor. The ectodomain of human E-cadherin contains four potential Nglycosylation sites at Asn residues 554, 566, 618, and 633. We investigated the role of E-cadherin N-glycosylation in cell cycle progression by site-directed mutagenesis. We showed previously that all four potential N-glycosylation sites of E-cadherin w ere N-gly cosylated in human breast carcinoma MDA-MB-435 cells. Removal of N-glycan at Asn633 dramatically affected E-cadherin stability. In this study we showed that E-cadherin mutant missing N-glycans at AsnS54, Asn566 and Asn618 failed to induce cell cycle arrest in Gt phase and to suppress cell proliferation in comparison with wild-type E-cadherin. Moreover, N-glycans at Asn554 and Asn566, but not at Asn618, seemed to be indispensable for E-cadherin-mediated suppression of cell cycle progression. Removal of N-glycans at either Asn554 or Asn566 of E-cadherin was accompanied with the activation of the extracellular signal-regulated protein kinase signaling pathway. After treatment with PD98059, an inhibitor of the extracellular signal-regulated protein kinase signaling pathway, wild-type E-cadherin transfected MDA-MB-435 and E-cadherin N-glycosylation-deficient mutant transfected MDA-MB-435 cells had equivalent numbers of cells in G1 phase. These rmdings implied that N-glycosylation might be crucial for E-cadherin-mediated suppression of cell cycle progression.     

Differentiation of propeptide residues regulating the compartmentalization, maturation and activity of the broad-range phospholipase C of Listeria monocytogenes

The intracellular bacterial pathogen Listeria monocytogenes secretes a broad-range phospholipase C enzyme called PC-PLC (phosphatidylcholine phospholipase C) whose compartmentalization and enzymatic activity is regulated by a 24-amino-acid propeptide (Cys28-Ser51). During intracytosolic multiplication, bacteria accumulate the proform of PC-PLC at their membrane-cell-wall interface, whereas during cell-to-cell spread vacuolar acidification leads to maturation and rapid translocation of PC-PLC across the cell wall in a manner that is dependent on Mpl, the metalloprotease of Listeria. In the present study, we generated a series of propeptide mutants to determine the minimal requirement to prevent PC-PLC enzymatic activity and to identify residues regulating compartmentalization and maturation. We found that a single residue at position P1 (Ser51) of the cleavage site is sufficient to prevent enzymatic activity, which is consistent with P1' (Trp52) being located within the active-site pocket. We observed that mutants with deletions at the N-terminus, but not the C-terminus, of the propeptide are translocated across the cell wall more effectively than wild-type PC-PLC at a physiological pH, and that individual amino acid residues within the N-terminus influence Mpl-mediated maturation of PC-PLC at acidic pH. However, deletion of more than 75% of the propeptide was required to completely prevent Mpl-mediated maturation of PC-PLC. These results indicate that the N-terminus of the propeptide regulates PC-PLC compartmentalization and that specific residues within the N-terminus influence the ability of Mpl to mediate PC-PLC maturation, although a six-residue propeptide is sufficient for Mpl to mediate PC-PLC maturation.     

Isolation and characterization of a stable activation intermediate of the lysosomal aspartyl protease cathepsin D.

Procathepsin D is the intracellular aspartyl protease precursor of cathepsin D, a major lysosomal enzyme. Procathepsin D is rapidly processed inside the cell, and, thus, examination of its proteolyic activation and structure has been difficult. To study this proenzyme, a nonglycosylated form of the human fibroblast procathepsin D was expressed in Escherichia coli, refold in vitro, and purified by affinity chromatography on pepstatinyl agarose. Sequence analysis of the refolded, autoactivated enzyme allowed determination of the autoproteolytic cleavage site. The sequence surrounding this cleavage site between residues LeuP26 and IleP27 (in the "pro" region) resembled the first cleavage site found during activation of other aspartyl proteases. Thus, the autoactivated procathepsin D is analogous to the pepsin activation intermediate, which has been termed pseudopepsin. The enzymatic activity, thermal and pH stability, and fluorescence spectra of pseudocathepsin D were compared to mature, predominantly two-chain, cathepsin D isolated from human placenta. The results indicated that pseudocathepsin D and mature enzyme have a similar Km toward a peptide substrate and cleave a protein substrate at identical sites. Temperature stability of the recombinant enzyme was similar to that of the tissue-derived enzyme. However, the recombinant enzyme had increased stability at low pH when compared to the glycosylated tissue-derived two-chain cathepsin D. Fluorescence spectra of the recombinant and tissue-derived enzymes were identical. Thus, the absence of asparagine-linked oligosaccharides and the presence of the remaining segment of propeptide did not significantly alter the structural and enzymatic properties of the enzyme.     

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.     

The role of the cathepsin E propeptide in correct folding, maturation and sorting to the endosome

Cathepsin E (CE) is an endosomal aspartic proteinase of the A1 family that is highly homologous to the lysosomal aspartic proteinase cathepsin D (CD). Newly synthesized CE undergoes several proteolytic processing events to yield mature CE, from which the N-terminal propeptide usually comprising 39 amino acids is removed. To define the role of the propeptide of CE in its biosynthesis and processing, we constructed two fusion proteins using chimeric DNAs encoding the CE propeptide fused to the mature CD tagged with HA at the COOH terminus (termed ED-HA) and encoding the CD propeptide fused to the mature CE (termed DE). Pulse-chase analysis revealed that wild-type CE expressed in human embryonic kidney cells is autoproteolytically processed into mature CE within a 12-h chase, whereas the chimeric DE failed to be converted into mature CE even after a 24-h chase. The DE chimera was nevertheless capable of acid-dependent autoactivation in vitro to yield a catalytically active form, although its specificity constants (kcat/Km) were considerably high but less (35%) than those of the wild-type CE. By contrast, the chimeric ED-HA expressed in HeLa cells underwent neither processing into a catalytically active enzyme nor acid-dependent autoactivation in vitro. The ED-HA protein was less stable than wt-CD-HA, as determined on pulse-chase analysis and on trypsin digestion. These data indicate that the propeptide of CE is essential for the correct folding, maturation, and targeting of this protein to its final destination.     

N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.     

Mutational analysis of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is a member of an N-terminal nucleophile hydrolase enzyme superfamily, several of which undergo autocatalytic propeptide processing to generate the mature active enzyme. A series of mutations was analyzed to determine whether amino acid residues required for catalysis are also used for propeptide processing. Propeptide cleavage was strongly inhibited by replacement of the cysteine nucleophile and two residues of an oxyanion hole that are required for glutaminase function. However, significant propeptide processing was retained in a deletion mutant with multiple defects in catalysis that was devoid of enzyme activity. Intermolecular processing of noncleaved mutant enzyme subunits by active wild-type enzyme subunits was not detected in hetero-oligomers obtained from a coexpression experiment. While direct in vitro evidence for autocatalytic propeptide cleavage was not obtained, the results indicate that some but not all of the amino acid residues that have a role in catalysis are also needed for propeptide processing.     

N-glycosylation of the beta-propeller domain of the integrin alpha5 subunit is essential for alpha5beta1 heterodimerization, expression on the cell surface, and its biological function

The N-glycosylation of integrin alpha5beta1 is thought to play crucial roles in cell spreading, cell migration, ligand binding, and dimer formation, but the underlying mechanism remains unclear. To investigate the importance of the N-glycans of this integrin in detail, sequential site-directed mutagenesis was carried out to remove single or combined putative N-glycosylation sites on the alpha5 integrin. Removal of the putative N-glycosylation sites on the beta-propeller, Thigh, Calf-1, or Calf-2 domains of the alpha5 subunit resulted in a decrease in molecular weight compared with the wild type, suggesting that all of these domains contain attached N-glycans. Importantly, the absence of N-glycosylation sites (sites 1-5) on the beta-propeller resulted in the persistent association of integrin subunit with calnexin in the endoplasmic reticulum, which subsequently blocked heterodimerization and its expression on the cell surface. Interestingly, the activities for cell spreading and migration for the alpha5 subunit carrying only three potential N-glycosylation sites (3-5 sites) on the beta-propeller were comparable with those of the wild type. In contrast, mutation of these three sites resulted in a significant decrease in cell spreading as well as functional expression, although the total expression level of the Delta3-5 mutant on the cell surface was comparable with that of wild type. Furthermore, we found that site 5 is a most important site for its expression on the cell surface, whereas the S5 mutant did not show any biological functions. Taken together, this study reveals for the first time that the N-glycosylation on the beta-propeller domain of the alpha5 subunit is essential for heterodimerization and biological functions of alpha5beta1 integrin and might also be useful for studies of the molecular structure.     

Characterization of rat cathepsin E and mutants with changed active-site residues and lacking propeptides and N-glycosylation, expressed in human embryonic kidney 293T cells

To study the roles of the catalytic activity, propeptide, and N-glycosylation of the intracellular aspartic proteinase cathepsin E in biosynthesis, processing, and intracellular trafficking, we constructed various rat cathepsin E mutants in which active-site Asp residues were changed to Ala or which lacked propeptides and N-glycosylation. Wild-type cathepsin E expressed in human embryonic kidney 293T cells was mainly found in the LAMP-1-positive endosomal organelles, as determined by immunofluorescence microscopy. Consistently, pulse-chase analysis revealed that the initially synthesized pro-cathepsin E was processed to the mature enzyme within a 24 h chase. This process was completely inhibited by brefeldin A and bafilomycin A, indicating its transport from the endoplasmic reticulum (ER) to the endosomal acidic compartment. Mutants with Asp residues in the two active-site consensus motifs changed to Ala and lacking the propeptide (Leu23-Phe58) and the putative ER-retention sequence (Ser59-Asp98) were neither processed nor transported to the endosomal compartment. The mutant lacking the ER-retention sequence was rapidly degraded in the ER, indicating the importance of this sequence in correct folding. The single (N92Q or N324D) and double (N92Q/N324D) N-glycosylation-deficient mutants were neither processed into a mature form nor transported to the endosomal compartment, but were stably retained in the ER without degradation. These data indicate that the catalytic activity, propeptides, and N-glycosylation of this protein are all essential for its processing, maturation, and trafficking.     

Maturation of Pichia pastoris-derived recombinant pro-Der p 1 induced by deglycosylation and by the natural cysteine protease Der p 1 from house dust mite.

The mature cysteine protease from Dermatophgoides pteronyssinus, Der p 1, is a major house dust mite allergen. Its enzymatic activity has been shown to have pro-inflammatory effects that could also negatively influence efficacy of allergen-specific immunotherapy. The aim of this study was to express recombinant pro-Der p 1 (rpro-Der p 1) in the yeast Pichia pastoris and to study its maturation. Expression was achieved at a concentration ranging from 45 mg.L-1 (methanol-induced expression) to 168 mg.L-1 (constitutive expression). No significant spontaneous maturation of the secreted proenzyme was observed. rpro-Der p 1 with a sequence-based molecular mass of 34 kDa was hyperglycosylated by the yeast, migrating at 50-60 kDa on SDS/PAGE. Compared with its natural counterpart (nDer p 1), the recombinant proenzyme demonstrated decreased IgE reactivity, resulting in a 30-fold lower capacity to induce histamine release from human basophils. Decreased immunoreactivity was also shown by competitive RIA and sandwich ELISA with Der p 1-specific antibody reagents. CD spectra of rpro-Der p 1 and nDer p 1 revealed significant structural differences. Deglycosylation of rpro-Der p 1 with endoglycosidase H resulted in a decrease in apparent molecular mass from 50 kDa to 34 kDa, but did not affect nDer p 1. On removal of N-glycans from rpro-Der p 1, which harbours two putative N-glycosylation sites in both propeptide and mature sequence, the mature rDer p 1 appeared. This suggests that hyperglycosylation hampers spontaneous maturation. Maturation of the recombinant pro-enzyme was also achieved by addition of the active natural cysteine protease, nDer p 1. In conclusion, high-level expression of rpro-Der p 1 in P. pastoris results in a stable hypoallergenic proenzyme with potential for use in allergen-specific immunotherapy.     

Folding pathway mediated by an intramolecular chaperone: dissecting conformational changes coincident with autoprocessing and the role of Ca(2+) in subtilisin maturation.

Subtilisin is produced as a precursor that requires its N-terminal propeptide to chaperone the folding of its protease domain. Once folded, subtilisin adopts a remarkably stable conformation, which has been attributed to a high affinity Ca(2+) binding site. We investigated the role of the metal ligand in the maturation of pro-subtilisin, a process that involves folding, autoprocessing and partial degradation. Our results establish that although Ca(2+) ions can stabilize the protease domain, the folding and autoprocessing of pro-subtilisin take place independent of Ca(2+) ion. We demonstrate that the stabilizing effect of calcium is observed only after the completion of autoprocessing and that the metal ion appears to be responsible for shifting the folding equilibrium towards the native conformation in both mature subtilisin and the autoprocessed propeptide:subtilisin complex. Furthermore, the addition of active subtilisin to unautoprocessed pro-subtilisin in trans does not facilitate precursor maturation, but rather promotes rapid autodegradation. The primary cleavage site that initiates this autodegradation is at Gln19 in the N-terminus of mature subtilisin. This corresponds to the loop that links alpha-helix-2 and beta-strand-1 in mature subtilisin and has indirect effects on the formation of the Ca(2+) binding site. Our results show that the N-terminus of mature subtilisin undergoes rearrangement subsequent to propeptide autoprocessing. Since this structural change enhances the proteolytic stability of the precursor, our results suggest that the autoprocessing reaction must be completed before the release of active subtilisin in order to maximize folding efficiency.     

Importance of protease cleavage sites within and flanking human immunodeficiency virus type 1 transframe protein p6* for spatiotemporal regulation of protease activation

The human immunodeficiency virus type 1 (HIV-1) protease (PR) has recently been shown to be inhibited by its propeptide p6* in vitro. As p6* itself is a PR substrate, the primary goal of this study was to determine the importance of p6* cleavage for HIV-1 maturation and infectivity. For that purpose, short peptide variants mimicking proposed cleavage sites within and flanking p6* were designed and analyzed for qualitative and quantitative hydrolysis in vitro. Proviral clones comprising the selected cleavage site mutations were established and analyzed for Gag and Pol processing, virus maturation, and infectivity in cultured cells. Amino-terminal cleavage site mutation caused aberrant processing of nucleocapsid proteins and delayed replication kinetics. Blocking the internal cleavage site resulted in the utilization of a flanking site at a significantly decreased hydrolysis rate in vitro, which however did not affect Gag-Pol processing and viral replication. Although mutations blocking cleavage at the p6* carboxyl terminus yielded noninfectious virions exhibiting severe Gag processing defects, mutations retarding hydrolysis of this cleavage site neither seemed to impact viral infectivity and propagation in cultured cells nor seemed to interfere with overall maturation of released viruses. Interestingly, these mutants were shown to be clearly disadvantaged when challenged with wild-type virus in a dual competition assay. In sum, we conclude that p6* cleavage is absolutely essential to allow complete activation of the PR and subsequent processing of the viral precursors.     

Importance of the propeptide in the biosynthetic maturation of rat cathepsin C

Cathepsin C is a cysteine dipeptidyl-aminopeptidase. Active cathepsin C is found in lysosomes as a 200-kDa multimeric enzyme. Subunits constituting this assembly all arise from the proteolytic cleavage of a single precursor giving rise to three peptides: the propeptide, the alpha- and the beta-chains. Some features of the propeptide such as its length, its high level of glycosylation and its retention in the active lysosomal form of the enzyme suggest an important contribution of the proregion in the transport, maturation and expression of cathepsin C. In order to assess some aspects of this contribution, we transiently expressed mutant molecules of rat cathepsin C either lacking three of the four glycosylation sites, partially deleted in the proregion, or mutated at tryptophan 39 also located in the proregion, and studied their biosynthesis. Our results show that at least one of the three glycosylation sites in the propeptide must be glycosylated in order to obtain targeting and maturation of cathepsin C. We also show that a deletion of 14 amino acids and mutation W39S in the propeptide totally abolishes the biosynthetic processing of the enzyme. These results demonstrate that in addition to its role as a chaperone or in maintaining the latency of the enzymatic activity, the propeptide is required for proper transport and expression of newly synthesized cathepsin C.     

Influence of an ER-retention signal on the N-glycosylation of recombinant human α-l-iduronidase generated in seeds of Arabidopsis

Processes associated with late events of N-glycosylation within the plant Golgi complex are a major limitation to the use of plant-based systems to produce recombinant pharmaceutical proteins for parenteral administration. Specifically, sugars added to the N-glycans of a recombinant protein during glycan maturation to complex forms (e.g. β1,2 xylose and α1,3 fucose) can render the product immunogenic. In order to avoid these sugars, the human enzyme α-L-iduronidase (IDUA, EC 3.2.1.76), with a C-terminal ER-retention sequence SEKDEL, was expressed in seeds of complex-glycan-deficient (cgl) mutant and wild-type (Col-0) Arabidopsis thaliana, under the control of regulatory (5'-, signal-peptide-encoding-, and 3'-) sequences from the arcelin 5-I gene of Phaseolus vulgaris (cgl-IDUA-SEKDEL and Col-IDUA-SEKDEL, respectively). The SEKDEL motif had no adverse effect on the specific activity of the purified enzyme. Surprisingly, the majority of the N-glycans of Col-IDUA-SEKDEL were complex N-glycans (i.e. contained xylose and/or fucose) (88 %), whereas complex N-glycans comprised a much lower proportion of the N-glycans of cgl-IDUA-SEKDEL (26 %), in which high-mannose forms were predominant. In contrast to the non-chimeric IDUA of cgl seeds, which is mainly secreted into the extracellular spaces, the addition of the SEKDEL sequence to human recombinant IDUA expressed in the same background led to retention of the protein in ER-derived vesicles/compartments and its partial localization in protein storage vacuoles. Our data support the contention that the use of a C-terminal ER retention motif as an effective strategy to prevent or reduce complex N-glycan formation, is protein specific.     

Clarification of the role of N-glycans on the common beta-subunit of the human IL-3, IL-5 and GM-CSF receptors and the murine IL-3 beta-receptor in ligand-binding and receptor activation

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 are related cytokines that play key roles in regulating the differentiation, proliferation, survival and activation of myeloid blood cells. The cell surface receptors for these cytokines are composed of cytokine-specific alpha-subunits and a common beta-receptor (betac), a shared subunit that is essential for receptor signaling in response to GM-CSF, IL-3 and IL-5. Previous studies have reached conflicting conclusions as to whether N-glycosylation of the betac-subunit is necessary for functional GM-CSF, IL-3 and IL-5 receptors. We sought to clarify whether betac N-glycosylation plays a role in receptor function, since all structural studies of human betac to date have utilized recombinant protein lacking N-glycosylation at Asn(328). Here, by eliminating individual N-glycans in human betac and the related murine homolog, beta(IL-3), we demonstrate unequivocally that ligand-binding and receptor activation are not critically dependent on individual N-glycosylation sites within the beta-subunit although the data do not preclude the possibility that N-glycans may exert some sort of fine control. These studies support the biological relevance of the X-ray crystal structures of the human betac domain 4 and the complete ectodomain, both of which lack N-glycosylation at Asn(328).