Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, CB#7248, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
Received on September 10, 2003; revised on October 29, 2003; accepted on November 27, 2003
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Abstract |
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Key words: C-mannosylation / mucins / mucin biosynthesis / MUC5AC / MUC5B
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Introduction |
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The predominant molecular forms of MUC5AC and MUC5B in mucus are thought to be monomers linked by interchain disulfide bonds to form linear and flexible oligomers/multimers (Hovenberg et al., 1996; Rose et al., 1984
; Slayter et al., 1984
; Thornton et al., 1990
; Wickstrom et al., 1998
). However, studies with porcine submaxillary mucin and MUC2 suggest also the existence of branched oligomers/multimers (Godl et al., 2002
; Perez-Vilar and Hill, 1999
). Each gel-forming mucin monomer consists of a predominant heavily O-glycosylated central region flanked at both sides by different cysteine-rich domains (Figure 1A). Some of the later domains, including D- and CK domains (Perez-Vilar, unpublished data), are involved in the assembly of disulfide-linked oligomers/multimers (Godl et al., 2002
; Perez-Vilar and Hill, 1999
). The central O-glycosylated regions in MUC5AC (Escande et al., 2001
), MUC5B (Desseyn et al., 1997
) and its murine counterpart, Muc5b (Escande et al., 2002
), are formed by threonine/serine-rich sequences, to which O-glycans are covalently linked, alternating with 110-residue-long, half-cysteine rich regions known as the Cys subdomains. Nine and seven homologous Cys subdomains are found scattered in the central region of MUC5AC (GenBank/EMBL accession numbers AJ298317, AJ2983118, AJ2983119 and AJ292079) and MUC5B (GenBank/EMBL accession number Z72496), respectively (Desseyn et al., 1997
; Escande et al., 2001
) (Figure 1A). Only the Cys subdomains show amino acid homologies between MUC5AC and MUC5B, whereas the Ser/Thr-rich domains have different amino acid sequences. Cys subdomains are present in other vertebrate gel-forming mucins, including MUC2, rMuc2, and porcine gastric mucin (Cao et al., 1999
; Perez-Vilar and Hill, 1999
). The high intra- and inter-species protein homologies suggest the Cys subdomains play critical but undefined roles in mucus homeostasis.
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Our early biochemical studies with recombinant mucin domains expressed in COS-7 cells have identified structural features that could play critical roles in the processing, assembly, and secretion of vertebrate gel-forming mucins (Perez-Vilar et al., 1996, 1998
; Perez-Vilar and Hill, 1998a
,b
, 1999
). This model system circumvents some of the difficulties associated with the high molecular weight, polymorphism, and extent of glycosylation of these glycoproteins for studying the function of unique mucin domains. Using this model system and also expression in Chinese hamster ovary (CHO) cells to explore mucin Cys subdomain function during mucin biosynthesis, we show here that MUC5AC and MUC5B Cys subdomains are C-mannosylated and that lack of C-mannosylation results in the arrest of these protein domains in the endoplasmic reticulum (ER).
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Results |
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As shown in Figure 4B, and consistent with studies shown in Figure 4A, monomers of the mucin Cys subdomains were the major species observed. MUC5AC/Cys5 monomers could be readily distinguished by SDSgel electrophoresis because the molecular weight of MUC5AC/Cys5 is higher than the molecular weight of MUC5AC/Cys1, MUC5B/Cys1, or MUC5B/Cys3 (see bottom insert in Figure 4B and also Figure 2B). In the same gels and in spite of the cross-linker employed, protein bands corresponding to dimers/oligomers of the Cys subdomains were barely detected and needed longer exposure times to be clearly identified, as shown in the full gel at the top of Figure 4B. Monomeric and dimeric but not oligomeric species of ECD/S-NHS-treated proteins (lanes 2, 4 and 6), migrated slightly slower in SDS gels than those cross-linked with BS3 (lanes 1, 3 and 5), probably as a result of differences in the extent of amino acid side chain modifications. These results indicate that if heterotypic interactions involving two different subdomains had occurred, these were very weak as for the homotypic interactions involving just one domain. Considered together, the cross-linking studies are inconsistent with the existence of strong, noncovalent, homo- or heterotypic interactions between Cys subdomains at the conditions tested.
C-mannosylation of mucin Cys subdomains
The presence of a WXXW amino acid sequence in the N-terminal side of all mucin Cys subdomains is one of their most distinctive features (Desseyn et al., 1997; Escande et al., 2001
) (Figures 1B and 1C). Because it has been recently shown that conserved WXXW sequences in extracellular proteins are acceptor motifs for protein C-mannosylation, by which a single mannose is bound to the first tryptophan in the motif (Krieg et al., 1997
), we initiated studies to test whether mucin Cys subdomains were C-mannosylated. The glycosyltransferase activity responsible for C-mannosylation has been detected in COS-7 cells (Krieg et al., 1997
); therefore this cell system was suitable for our purposes. However, attempts to detect mannosylation of MUC5AC/Cys5 or MUC5B/Cys3 by metabolic labeling of the protein using [3H]-mannose or [3H]-glucose were unsuccessful (data not shown). The fact that only a single [3H]-mannose can be added per mucin Cys subdomain, together with the low energy of the [3H]-mannose, the competition for this monosaccharide by other glycosylation processes, and the rather low level of protein expression obtained in our experimental system, provide a likely explanation for the lack of sensitivity of this approach.
As an alternative strategy, we used affinity chromatography with mannose-specific lectins as adsorbents of MUC5AC/Cys5 and MUC5B/Cys3. This method, though indirect and not of general application, seemed especially suitable for our case for two reasons. First, MUC5AC/Cys5 and MUC5B/Cys3 are not N-glycosylated and, based on their molecular weights, lack large carbohydrate chains. N-linked oligosaccharide chains are the major source of mannose, whereas O-linked oligosaccharide or glycosaminoglycan chains might interfere nonspecifically with the lectin-binding assays. Second, a lectin-based approach permitted analysis of low amounts of radioactive mucin domains. Galanthus nivalis agglutinin (GNA) (Hester and Wright, 1996) and Lens culinaris agglutinin (LCA) (Loris et al., 1994
), two structurally unrelated lectins that recognize nonreducing terminal mannose residues, were used for these studies.
Figure 5A shows that MUC5AC/Cys5 can be purified by lectin absorption using GNA-agarose beads. In these studies, COS-7 cells expressing MUC5AC/Cys5 were incubated with [35S]-amino acids and secreted proteins purified by different means and analyzed by gel electrophoresis. Metal-affinity absorption of the secreted proteins yielded one protein band of Mr 13,100 (lane 1) corresponding to MUC5AC/Cys5, which is consistent with results shown in Figure 2A. Direct incubation of the culture medium with GNA-agarose beads, however, showed the binding of many glycoproteins to the adsorbent, of which one with a Mr
13,100 was among the most abundant (lane 2). This protein band was not observed following lectin-affinity purification of culture media from cells transfected with the parental plasmid, pSecTag2A (data not shown). The 13,100 Mr protein could be repurified by metal-affinity absorption after it was eluted from the GNA-agarose resin with buffered 4 M guanidine-HCl (lane 3), which identified this protein as the His-tagged MUC5AC/Cys5. Consistent with these results, MUC5AC/Cys5 bound to and eluted from the metal-affinity adsorbant, could be repurified with GNA-agarose (lane 4). Binding to GNA-agarose was judged to be mannose-dependant based on the following observations: (1) the mucin Cys subdomains did not bind to uncoupled agarose (data not shown); (2) mucin Cys subdomains could be purified using another, structurally unrelated, mannose-specific lectin (LCA) (Furmanek and Hofsteenge, 2000
) (Figure 5B); and (3) binding of the Cys subdomain to the lectin adsorbent was substantially reduced in the presence of free mannose in the binding buffer (Figure 5C). These results suggest mucin Cys subdomains were mannosylated during their biosynthesis in transfected cells.
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Unmannosylated mucin Cys subdomains are retained in the ER
Figure 8A shows the results from experiments in which COS-7 cells were transfected with three independent clones for MUC5AC/Cys5 or MUC5AC/Cys5(W/A), respectively, and proteins in the medium absorbed to metal-affinity resin and analyzed by SDSgel electrophoresis. In all three cases, more MUC5AC/Cys5 (lanes 13) was recovered from the culture medium than was MUC5AC/Cys5(W/A) (lanes 46). This possibility was further supported by expression of mutant MUC5B/Cys3(W/A) of the MUC5B/Cys3 subdomain, in which the WXXW motif was changed by site-directed mutagenesis to AXXW. Only very low amounts of MUC5B/Cys3 were recovered from the culture medium of transfected cells (not shown). These observations could not be attributed to differences in the transcriptional/ translational efficiencies between the parental and mutated sequences because both types of constructs yield similar protein amounts in coupled in vitro transcription/translation assays (data not shown). These results suggest that the Cys subdomains lacking the C-mannosylation acceptor motif are poorly secreted.
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To determine the intracellular fate of the nonmannosylated Cys subdomains, the Cys subdomains were fused to the C-terminal end of green fluorescent protein (GFP), itself fused to the C-terminal end of a signal peptide, and the trafficking of the corresponding proteins assessed by GFP methods in live CHO-K1 or CHO-Lec35.1 cells. Preliminary experiments indicated that GFP-mucin fusion proteins were secreted from COS-7 and CHO-K1 cells but in lesser amounts from CHO-Lec35.1 cells, showing that GFP did not alter the secretion pattern of the mucin Cys subdomains. Figure 9A shows confocal images of live cells 24 h after transfection with plasmids encoding secreted GFP, named SHGFP (a and d) or the GFP-fusion proteins SHGFP-MUC5AC/Cys5 (b and e) and SHGFP-MUC5B/Cys3 (c and f), respectively. In CHO-K1 (a) and CHO-Lec35.1 cells (d), SHGFP was predominantly found in perinuclear tubulovesicular structures characteristic of the Golgi complex, very likely representing proteins in their way to secretion. These results show that the intracellular trafficking of SHGFP is not affected by the phenotype, including the lack of protein C-mannosylation, of CHO-Lec35.1 cells. This is not unexpected considering that in nature native GFP is not a secreted protein and, accordingly, does not have N-linked oligosaccharides or C-mannoses.
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Discussion |
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Rheological studies suggest mucus is formed by a mucin network held together by weak noncovalent bonds with the likely intervention of certain ions (Bansil et al., 1995; Madsen et al., 1998
; Raynal et al., 2003
), although other studies support the view that mucus viscoelastic properties are determined primarily by entanglement of the mucin oligomers/multimers (Carlstedt et al., 1985
; Verdugo, 1990
). Moreover, early reports documented the tendency of human tracheobronchial mucins, likely a mixture of MUC5AC and MUC5B, to form aggregates at low salt concentrations (Rose et al., 1984
; Slayter et al., 1984
), and recent investigations suggest that aggregation of respiratory mucins is caused by interchain hydrophobic interactions (Bromberg and Barr, 2000
). Furthermore, noncovalent interactions are thought to be critical during the packing of mucin precursors inside mucus granules of goblet/mucous cells. The specific roles that the different protein domains in mucins play in these interactions have not been defined yet. Interestingly, the Cys subdomains in porcine gastric mucin have been proposed to mediate hydrophobic proteinprotein interactions critical for mucus gel formation at low pH (Cao et al., 1999
). Our cross-linking studies, however, argued against the existence of strong noncovalent homo- and heterotypic interactions among the mucin Cys subdomains at neutral or slightly acidic pH, and only very weak noncovalent interactions were revealed using cross-linkers (Figure 4). The biological significance (if any) of these very weak interactions is not clear at present. Unfortunately, the amounts of expressed Cys subdomains in our cell system are unsuitable for using alternative, more powerful and precise binding techniques. In any case, it is tempting to speculate that in vivo the Cys subdomains might be protein regions where disulfide-linked oligomers/ multimers of MUC5AC or MUC5B contact one another, especially when certain conditions, for example, regarding pH and mucin concentration, are met.
MUC5AC and MUC5B, like others, are highly glycosylated with N- and especially O-linked oligosaccharides. Glycosylation is fundamental to their ability to form the mucus gels, which underlie the mucociliary clearance mechanism, and to interact with many different compounds. The present studies suggest that a third type of glycosylation, C-mannosylation, is also present in MUC5AC and MUC5B. Thus mucin Cys subdomains are secreted from transiently transfected cells as C-mannosylated proteins, based on the observations that MUC5AC/Cys5 and MUC5B/Cys3 bind to the mannose-specific lectins, GNA and LCA (Figure 5) and that this binding depends on the C-mannosylation recognition sequence WXXW in these domains (Figure 6). The absence of N-glycosylation recognition sites in MUC5AC/Cys5 and MUC5B/Cys3, eliminating the major source of mannose residues in mammalian glycoproteins, made feasible the use of mannose-specific lectins to reveal C-mannosylation of this domain. Moreover, the apparent molecular weights of these Cys subdomains were consistent with unmodified or under glycosylated proteins (Figure 2B), excluding the possibility of nonspecific interactions between the GNA or LCA lectins and O-linked oligosaccharide or glycosaminoglycan chains. Furthermore, other forms of protein glycosylation that add single monosaccharides (Spiro, 2002) and therefore could interfere with our lectin assay, seemed highly unlikely to occur in the mucin Cys subdomains. Thus N-acetyl-glucosamine-Ser/Thr linkages are not present in secretory proteins and both fucose-Ser/Thr and glucose-Ser linkages are primarily found in epidermal growth factorlike domains (Spiro, 2002
), which are protein domains structurally unrelated to the mucin Cys subdomains (Desseyn et al., 1997
; Escande et al., 2001
).
The change of the WXXW motif in MUC5AC/Cys5 to an AXXW sequence prevented binding of the domain to either LCA or GNA (Figure 6). It has been demonstrated that WXXW motifs in C-mannosylated proteins are the major acceptor sequences for the glycosyltransferase involved in this kind of glycosylation (Krieg et al., 1998; Doucey et al., 1999
). Therefore the binding of GNA or LCA to the Cys subdomains can be explained by the fact that this protein domain is C-mannosylated in its WXXW motif. This conclusion is supported by studies suggesting the primary (rather than tertiary) structure determines whether a protein is C-mannosylated. Thus the sequence TWAQWFET in RNAse2 has all the information required for C-mannosylation of this protein (Doucey et al., 1998
). The corresponding sequence in MUC5AC/Cys5, TWTTWFDV (Figure 1B and 1C), is very similar, strongly suggesting that the same structural information that determines C-mannosylation of Rnase2 is conserved in MUC5AC/Cys5 and likely in all mucin Cys subdomains (Figure 1C). Considered together, these observations suggest that the mucin Cys subdomains in MUC5AC and MUC5B are C-mannosylated in their respective WXXW motifs. The question arises whether C-mannosylation occurs in airway goblet/mucous cells. Unfortunately, neither the enzyme responsible for this modification nor its gene has been characterized; therefore we are unable to confirm its presence in particular cell types. However, the current evidence strongly suggest C-mannosylation, like O- and N-glycosylation, is a widespread modification that has been found in all tissue and animal species tested (Furmanek and Hofsteenge, 2000
), including the lung (Hofsteenge, personal communication).
Our attempts to demonstrate or deny C-mannosylation in native MUC5AC and MUC5B have failed. Thus in these studies, which involve the generation, fractionation, and direct mass spectrometry sequencing of tryptic peptides derived from purified airways MUC5AC/MUC5B mucins, we have identified a set of peptides representative of many of the protein regions in the mucin Cys subdomains. Peptides located at the N-terminal side of the mucin Cys subdomains, where the WXXW motifs are located, were not found (Kesimer et al., unpublished data). The proximity of O-glycosylated, Ser/Thr-rich regions at the N-terminal side of the Cys subdomains, and therefore close to their respective N-terminal WXXW motifs, may have prevented the isolation or identification of these peptides, although alternative strategies are in progress. In any case, so far these results suggest native Cys subdomains, like their recombinant counterparts (Figure 2), are not modified with N- and O-linked oligosaccharides, glycosaminoglycan chains, or other major modification, supporting the view the recombinant Cys subdomains expressed in COS-7/CHO cells are valid model systems of their respective native domains.
The glycosyltransferase responsible for C-mannosylation has not yet been characterized, but it seems to be located in microsomal fractions and requires dolichol-P-mannose as donor of mannose (Anand et al., 2001; Doucey et al., 1998
), a compound synthesized in the ER, which suggests this organelle is the intracellular site for protein C-mannosylation. Our pulse-chase studies showed that C-mannosylation of MUC5AC/Cys5 occurred very rapidly and was not affected by the presence of brefeldin A (Figure 7), a compound that disorganizes the Golgi complex. These data are consistent with the conclusion that protein C-mannosylation takes place in the ER. Moreover, as will be discussed, lack of C-mannosylaton prevents transport of the Cys subdomains out of the ER. Therefore C-mannosylation, together with N-glycosylation and covalent dimerization via the CK domains (Perez-Vilar and Hill, 1999
; Asker et al., 1998a
,b
; Perez-Vilar unpublished data), would be the three major modifications of the MUC5AC and MUC5B polypeptides in the ER.
Although the role of protein C-mannosylation is unclear at present, our studies clearly suggest this modification is required for proper transport of the Cys subdomain out of the ER. Thus Cys subdomains with mutated WXXW motifs were poorly secreted from COS-7 cells (Figure 8A), whereas their normal counterparts were arrested inside the ER only when expressed in C-mannosylation defective cells (Figures 8B and 9A). It is well documented that misfolded or partially folded proteins are retained in the ER by the quality control mechanism that operates in this organelle (Ellgaard and Helenius, 2003). Hence, a critical role of C-mannosylation during mucin Cys subdomain folding would explain that in its absence the domains are retained inside the ER. There are three lines of indirect evidence supporting such a role for this modification. First, structural studies with recombinant eosinophil-derived neurotoxin, a C-mannosylated RNase, indicated C-mannosylation stabilizes its N-terminal loop, likely by keeping the first tryptophan in the WXXW motif in a specific orientation (Loffler et al., 1996
; Vliegenthart and Casset, 1998
). Both features are lost in the unglycosylated counterpart of this protein. Second, mutation of the conserved WXXW motif in the erythropoietin receptor arrests the protein in the ER (Hilton et al., 1996
), although whether the receptor was C-mannosylated was not studied. Third, experiments with RNase2 suggest C-mannosylation occurs prior to complete protein folding (Krieg et al., 1998
), suggesting this modification is cotranslational and hence able to influence folding of the nascent polypeptide. It is tempting to speculate that C-mannosylation of the Cys subdomains together with N-glycosylation of the D, C, and CK domains, cooperate with complementary mechanisms to allow correct folding of MUC5AC and MUC5B in the ER.
Alternatively, C-mannosylation might be part of a mechanism that regulates mucin ER export once mucin precursors are folded and ready to exit the organelle. It is increasingly clear that although some proteins seem to exit the ER by a bulk flow pathway, others are packaged into transport vesicles (Glick, 2001; Gorelick and Shugrue, 2001
). This packaging requires interactions between proteins in transit and vesicular coat subunits and/or cargo receptors. Thus C-mannoses might be involved in lectin-type interactions critical for mucin packaging at ER exit sites. In the ER, mucin precursors are very likely not O-glycosylated, a modification that largely occurs in the Golgi complex (Perez-Vilar and Hill, 1999
); therefore the Cys subdomains and their C-mannoses would be accessible to interact with ER proteins.
In conclusion, our studies suggest that a third type of protein glycosylation, C-mannosylation, exists in MUC5AC and MUC5B. C-mannoses would be located in the WXXW motifs at the N-terminal side of the Cys subdomains, which are interdispersed among the O-glycosylated regions of these mucins. C-mannosylation seems to be required during the early stages of mucin biosynthesis, either for the folding of the domain or for some aspect of the transport out of the ER.
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Materials and methods |
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Site-directed mutagenesis
Mutations were made as described (Deng and Nickoloff, 1992) with reagents from Clontech, the pSecTag2A-derived expression vectors as templates, and the following selection oligonucleotides (MGW Biotech.): 5'-CCCAAGGGCGAACTCTGCAGATATCC-3', which destroys a unique EcoRI site present in the nontranslated 3'-region of the expression vectors. The mutagenic oligonucleotides (MGW Biotech.) used to change the codon for tryptophan 1 to an alanine codon in the unique WXXW-encoding DNA sequences of pMUC5AC/Cys5 and pMUC5B/Cys3 were 5'-CCCCGGTGCACCGCGACAACGTGGTTCGT-3' and 5'-CAACCGAAGTGTGAGGCGACAGAGTGGTTTGACG-3', respectively. The complete DNA sequences encoding the respective mucin Cys subdomains were verified by DNA sequencing. The transcriptional/translational efficiencies of these two plasmids were assessed by coupled in vitro transcription/translation assays using the T7 TNT system from Promega (Madison, WI) as directed by the manufacturer. 35S-proteins were then analyzed by standard SDSpolyacrylamide gel electrophoresis and autoradiography with or without previous purification of the mucin domains with TALON-IMAC beads.
Transfection and analysis of recombinant proteins
CHO-K1 and CHO-Lec35.1 cells (kindly provided by Dr. M. A. Lehrman, Texas South Western Medical Center) where maintained in HAMF12 media containing 10% fetal bovine serum (FBS) whereas COS-7 cells were grown in Dulbecco's modified Eagle medium containing 10% FBS. Cells in 25-cm2 flasks were transfected with Fugene-6 (Roche, Indianapolis, IN) as described (Perez-Vilar and Hill, 1997). At 24 h posttransfection, cells were radiolabeled with [35S]-cysteine/methionine as reported previously (Perez-Vilar et al., 1996
) except that [35S]-Pro-mix (Amersham Biotech, Little Chalfont, United Kingdom) was used as a source of radiolabeled amino acids. Absorption of His-tagged proteins to TALON-IMAC beads (Clontech) was performed as described (Perez-Vilar and Hill, 1997
).
For purification of proteins with mannose-specific lectins, the culture media was cleared by centrifugation and mixed with an equal volume of 20 mM sodium phosphate, pH 7.2, 300 mM NaCl. The proteins where then absorbed to GNA or LCA lectin conjugated to agarose beads (both from Sigma, St. Louis, MO) (50 mg lectin-agarose, preequilibrated in phosphate buffered saline [PBS] [10 mM sodium phosphate, pH 7.2, 150 mM NaCl] per 2 ml diluted culture medium) for 30 min at 25°C under agitation. The beads were thoroughly washed in PBS; then 50 mM sodium phosphate, pH 7.2, 750 mM NaCl; and, finally, 10 mM TrisHCl, pH 7.5, prior to boiling the samples in SDSgel electrophoresis sample buffer. In some experiments, [35S]-proteins bound to lectin-agarose beads were eluted in 0.5 ml 4 M guanidine-HCl in 50 mM TrisHCl, pH 8.0, and subsequently absorbed to TALON-IMAC beads (20 mg/ml). In other experiments, His-tagged [35S]-proteins bound to TALON-IMAC beads were eluted with PBS containing 10 mM EDTA and absorbed to LCA or GNA-agarose beads (10 mg lectin-agarose per 0.5 ml eluate) as described.
Analysis of the proteins by SDSgel electrophoresis in Tris/glycine or Tris/tricine gels and autoradiography were done using standard methods. 14C-methylated proteins (Amersham) used as low molecular weight standards were carbonic anhydrase (30,000), trypsin inhibitor (21,500), cytochrome c (12,500), aprotinin (6500), and insulin (5740). Higher molecular weight standards were 14C-methylated myosin (220,000), phosphorylase b (97,400100,000), bovine serum albumin (66,000), ovalbumin (46,00050,000), carbonic anhydrase (30,000), and lysozyme (14,300).
Cross-linking studies
Cross-linking of proteins, previously absorbed to TALON-IMAC beads, with BS3 or EDC/S-NHS, all from Pierce (Rockford, IL), were performed essentially as described earlier for BS3 (Perez-Vilar et al., 1996) except that EDC/S-NHS cross-linking was carried out with 1 mM each in 0.1 M 2-(N-morpholino)ethanesulfonic acid, 0.9 % (w/v) NaCl, pH 6.0. For cross-linking with glutaraldehyde, His-tagged proteins bound to TALON IMAC beads were directly reacted with freshly prepared glutaraldehyde (0.010.1%, v/v; Sigma) in PBS for 0.54 h at 25°C in the presence or absence of 1 µg/ml bovine serum albumin (Sigma). In some experiments, purified proteins were cross-linked in the presence of 10 mM EDTA, which released proteins from the IMAC resin, or alternatively secreted proteins were cross-linked with BS3 before IMAC purification as described earlier (Perez-Vilar et al., 1996
).
Live cell imaging
CHO-Lec35.1 cells were subcultured in 35-mm glass-bottom dishes (MatTek, Ashland, MA) and 24 h later transfected with 1 µg DNA and 3 µl Fugene-6 as directed by the manufacturer. After 24 h, the culture medium was changed and the cells incubated for an additional hour. The medium was then replaced with phenol redfree, bicarbonate-free Dulbecco's modified Eagle medium, 5% FBS, 20 mM HEPES, and observed in a Zeiss LSM 510 (UNC Hooker Microscopy Facility, Chapel Hill, NC) at 37°C in the microscope stage using 488 nm laser excitation for GFP and 413 nm for CFP. Cells clones stably expressing the Cys subdomains were observed after seeding them in the same dishes and once they were 7585% at confluency. Images were analyzed using Zeiss LSM software.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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