2 Structural Glycobiology Section and 3 Intramural Research Support Program-SAIC, Laboratory of Experimental and Computational Biology, NCI-CCR, Building 469, Room 221, Frederick, MD, 21702-1201, USA
Received on December 6, 2001; revised on February 6, 2002; accepted on February 24, 2002
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Abstract |
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Key words: aspartate motifs/bovine ß4Gal-T1/catalytic domain/metal-binding sites/substrate binding
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Introduction |
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Although the bovine ß4Gal-T1 was the first glycosyltransferase to be isolated and cloned (Narimatsu et al., 1986; D'Agostaro et al., 1989
; Shaper et al., 1986
; Masibay and Qasba, 1989
; Russo et al., 1990
), it was not until recently that a family (ß4Gal-T2 to ß4Gal-T7) of related human and mouse ß4Gal-Ts was identified (Lo et al., 1998
; Amado et al., 1999
). These glycosyltransferases differ in the oligosaccharide acceptor specificities. Only ß4Gal-T1 and ß4Gal-T2 have lactose synthase activity in the presence of LA (Almeida et al., 1997
). ß4Gal-T4 has a weak interaction with LA and is involved in the synthesis of glycosphingolipids (Schwientek et al., 1998
). ß4Gal-T5 has 37% sequence identity to human ß4Gal-T1 but has no lactose synthase activity in the presence of LA (Sato et al., 1998a
,b). ß4Gal-T6, which has a 94% sequence identity with the rat lactosylceramide synthase and transfers galactose from UDP-Gal to glucosylceramide, shows 39% sequence identity to the catalytic domain of the mouse ß4Gal-T1 (Nomura et al., 1998
). The newly discovered ß4Gal-T7 shows 38% sequence identity to the Caenorhabditis elegans sqv-3 gene (Okajima et al., 1999
) and is reported to be involved in the synthesis of proteoglycans (Okajima et al., 1999
; Almeida et al., 1999
).
The specificities of glycosyltransferases are determined not only with respect to donor and acceptor substrates but also with regard to the nature of the glycosidic linkage ( or ß) that they form. The ß4Gal-T family members are classified as "inverting" enzymes because they transfer galactose from UDP-
-D-galactose to GlcNAc with an "inversion" of the configuration at the anomeric carbon atom of galactose, generating a ß1-4-linked product (Sinnott, 1990
). Bovine milk ß4Gal-T1 requires Mn2+ for the enzymatic reaction (Morrison and Ebner, 1971b
; Khatra et al., 1974
; Powell and Brew, 1974
; Bell et al., 1976
). Kinetic studies showed that the enzyme has two metal binding sites and that the occupation of both sites is required for maximum activation (Powell and Brew, 1976
). One of the sites binds Mn2+ with high affinity but does not bind Ca2+. The other is a low-affinity site that can also bind Ca2+ (Powell and Brew, 1976
; O'Keeffe et al., 1980
). Investigations of the metal ion requirements of the enzyme have shown that Zn2+, Cd2+, Fe2+, and Co2+ can also induce activation with lower activities at saturation than that of Mn2+. The DXD motifs, conserved in the family members of the glycosyltransferases (Breton et al., 1998
; Yuan et al., 1997
; Boeggeman and Qasba, 1998
; Wiggins and Munro, 1998
; Busch et al., 1998
; Shibayama et al., 1998
, 1999; Zhang et al., 1999
; Hodson et al., 2000
; Unligil et al., 2000
), have been shown to participate in metal binding and catalysis (Boeggeman and Qasba, 1998
; Wiggins and Munro, 1998
; Busch et al., 1998
; Shibayama et al., 1999
; Zhang et al., 1999
; Hodson et al., 2000
; Unligil et al., 2000
).
In the catalytic domain of the ß4Gal-T family members, two sequence regions, D242YDYNCFVFSDVD254 (region I) and W312GWGGEDDD320 (region II), contain the DXD motifs. The D242YD244 in region I is not a conserved DXD motif in all the ß4Gal-T family members; on the other hand, D252VD254 and E317DDD320 are highly conserved motifs. The kinetic mechanism of ß4Gal-T1 has been suggested to be an equilibrium-ordered mechanism, where UDP-Gal and Mn2+ form a complex with the enzyme prior to the binding of the sugar acceptor (Khatra et al., 1974; Powell and Brew, 1974
). It has also been suggested that the binding of the substrates to the enzymeMn2+ complex occurs via a random equilibrium mechanism (Bell et al., 1976
). The interaction of the enzymeMn2+ complex with UDP-Gal has been studied by circular dichroism, and the data indicate that a conformational change occurs on the binding of UDP-Gal to the enzyme-Mn2+ complex (Geren et al., 1975
) protecting one Trp residue from UV lightinduced destruction (Clymer et al., 1976
).
Although the crystal structure of the catalytic domain of bovine ß4Gal-T1, with and without UDP, has been determined at a 2.4 Å resolution (Gastinel et al., 1999), it did not provide the exact location of the sugar acceptor or that of Mn2+. In our laboratory, we have determined the crystal structure of the complexes of bovine ß4Gal-T1 and mouse
-LA in the presence of Mn2+ with various acceptor substrates, at 2 Å resolution (Ramakrishnan and Qasba, 2001
). These show that the Mn2+ ion is coordinated with five atoms; two of these are the pyrophosphate ionic oxygen atoms of the sugar nucleotide, and the remaining three atoms are the C
of Asp254, the N
2 atom of His347, and the S
atom of Met344 of ß4Gal-T1. The structures reveal a conformational change to have occurred within the region comprising residues 345365 of ß4Gal-T1 from the structure obtained by Gastinel et al. (1999)
(conformation I) to the structure (conformation II) we found (Ramakrishnan and Qasba, 2001
). This change positions His347 of the region in such a way that it can bind the Mn2+ ion, which subsequently can bind to the phosphates of the UDP-Gal. The second metal binding site was never identified in the LS, UDP-Gal and Mn2+ complex even though 17 mM MnCl2 was used during crystallization. The sequence region II, W312GWGGEDDD320, has a loop structure that is similar in conformations, I and II, with subtle differences. In conformation II, the side chain orientation of Trp314 allows it to interact with GlcNAc. Asp318 and Asp319 of the E317DDD320 sequence motif, localized in the loop structure, are also involved in GlcNAc binding. Asp320 does not appear to directly bind GlcNAc (Ramakrishnan and Qasba, 2001
).
In the present study, using site-directed mutagenesis we investigated the involvement of residues Asp242, Asp244, Asp252, Asp254, His347, and Met344 in metal-ion binding and catalysis. Mutation of any of these residues, except Asp242 and Asp244, affect the activity of the enzyme. The kinetic properties of the wild-type and mutated ß4Gal-T1 show that among the residues Asp254, His347, and Met344, which coordinate Mn2+ (Ramakrishnan and Qasba, 2001), only Asp254 and His347 are important for metal binding. Met344 can be replaced with alanine or glutamine with only a partial loss of enzyme activity. In addition, Glu317 and Asp320 mutants show an effect on their activities that is opposite to the wild-type enzyme when partially activated by Mn2+ and subsequently treated with Ca2+ or Co2+ ions.
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Results |
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Involvement of residues Asp254, Met344 and His347 in metal binding
In the absence of metal ions, ß4Gal-T1 does not catalyze the transfer of galactose from UDP-Gal to GlcNAc. The structure of ß4Gal-T1·UDP·LA·Mn2+ (Ramakrishnan and Qasba, 2001) showed that of the two Asp residues of the D252VD254 motif, only the carboxylate oxygen of Asp254 coordinates with Mn2+. On the other hand, Asp252 binds only to the galactose moiety of UDP-Gal in the donor substrate binding pocket of ß4Gal-T1 (Ramakrishnan et al., 2002
). Also, in the ß4Gal-T1·UDP-Glc·LA·Mn2+ complex, Asp252 binds to the glucose moiety of UDP-Glc (Ramakrishnan et al., 2001
). Although the mutation of Asp252 completely abolishes the catalytic activity of the enzyme, the mutant D252N binds to an UDP-agarose column like the enzymatically active wild type. Also, the mutants of Asp254, D254E and D254N barely retain 0.01% of the catalytic activity of ß4Gal-T1 (Table I). Due to the low levels of activity, the kinetic parameters for these mutants could not be obtained with confidence. The results are consistent with the finding that these residues, Asp252 and Asp254, are highly conserved among all the members of the ß4Gal-T family (Breton et al., 1998
).
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Effect of Mn2+ ion on the enzymatic activity of Met344 mutants
Enzyme activities of the wild-type and the Met344 mutants were measured at Mn2+ concentrations from 0 to 5 mM and at fixed concentrations of GlcNAc and UDP-Gal. The Mn2+ iondependent activation of M344A and M344Q showed a sigmoidal behavior (Figure 1). At least 20 µM Mn2+ is required to detect the activation of Met mutants, compared to about 4 µM for the wild-type enzyme. The mutants clearly require a higher Mn2+ concentration for activation than the wild-type protein (Figure 1). The enzymatic activities of ß4Gal-T1 and M344A at Mn2+ concentrations from 0 to 5 mM fitted well to a rate equation describing two metal binding sites, I (high affinity) and II (low affinity). Mutant M344A, compared to the wild-type, shows a fourfold lower binding affinity for site I and no effect on the affinity for site II (Table II). The data for M344Q did not fit well to Equation 3 (see Materials and methods), which describes two metal binding sites. However, the rate equation for a single metal binding site, Equation 4 (see Materials and methods), gave a better fit (Table II), showing higher affinity for Mn2+ but with a sevenfold reduction in the associated velocity.
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Effect of Co2+ and Ca2+ at a low Mn2+ concentration on the activation of Glu317 and Asp320
It has been shown previously that at low Mn2+ ion concentrations, Co2+ inhibits the activity of milk ß4Gal-T1 (Powell and Brew, 1976). In the presence of a fixed Mn2+ concentration (20 µM), the recombinant ß4Gal-T1 is inhibited with increasing concentrations of Co2+ behaving exactly like milk ß4Gal-T1 (Figure 5A). Displacement of Mn2+ by Co2+ in the wild-type enzyme produces an enzymemetal complex with lower activity causing enzyme inhibition (Powell and Brew, 1976
). With increasing concentrations of Co2+, Mn2+ is displaced from the high-affinity binding site, thus inhibiting the enzyme (Figure 5A). In strong contrast, Co2+ activates the mutants D320E, D320N, and E317D at 100 µM Mn2+ (Figure 5A). These results indicate that Co2+ is a more effective cofactor for these mutants compared to wild-type ß4Gal-T1.
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Kinetic parameters for GlcNAc and UDP-Gal for Glu317 and Asp320 mutants in the presence of Mn2+
The initial velocity data for ß4Gal-T1 and for the mutants were obtained at saturating concentrations of Mn2+ (10 mM) with varying concentrations of UDP-Gal and a series of fixed concentrations of GlcNAc (Table VI). The data were analyzed using the equations (see Materials and methods) that represent the sequential symmetrical initial velocity pattern (Equation 1) or the asymmetric initial velocity pattern (Equation 2). The data for the mutant E317D did not fit well to Equation 1, which is associated with an ordered or random equilibrium mechanism. Equation 2, associated with a ping-pong mechanism, gave a better fit where the value for Ki was zero. The Km for GlcNAc does not change in the mutant E317D, whereas the Km for UDP-Gal increases 2-fold compared to ß4Gal-T1 (Table VI). Using Eq. 1, the true Km values for UDP-Gal (Kmd) and GlcNAc (Kma) were determined for the D320 mutants and compared to the wild type. The Km for UDP-galactose increased fivefold for D320N, but it decreased to a third in the mutant D320E (Table VI). The equilibrium dissociation constant, Kid, for UDP-Gal increased by about 1.5- to 3-fold in these mutants. The Km for GlcNAc for the mutants D320E and D320N, compared to the wild type, showed values 11- and 151-fold higher, respectively. The turnover number, kcat, however, was reduced, resulting in a 240- to 700-fold decrease in the catalytic efficiency (kcat/Kma * Kid). kcat, can be influenced by both the rate-limiting acceptor binding and the product release steps; therefore, the specificity constants (kcat/Kmd * Kia or kcat/Kma * Kid), which combine rate and binding, are a measure of the stability of the transition state complex. Thus, extending the side chain length of Asp320 indirectly affects the binding of the acceptor and the binding of UDP-Gal to a lesser degree. It also affects the interaction between the enzyme and the substrate in the transition state during the catalytic mechanism.
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Discussion |
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The E317DDD320 sequence localized in the conserved sequence W312GWGGEDDD320 (region II) also contains the DXD motif, which is a potential site for the coordination of divalent cations (Boeggeman and Qasba, 1998). Substituting Asp318 or Asp319 with either asparagine or alanine or glutamic acid almost totally abrogated the activity (Boeggeman and Qasba, 1998
) (Table IV), and it was not possible to analyze the kinetic parameters for these mutants. The results presented by Zhang et al. (1999)
also show that mutating Asp318 with either asparagine or glutamic acid results in 0.1% of the activity of the wild-type protein. This region in the two structures, conformation I and II, also shows differences as evidenced by the side chain orientation of Trp314 (Figures 7A and 7B). In conformation II, the orientation of the side chain of Trp314 is such that it can make hydrophobic interactions with GlcNAc (Figure 7B), as well as with the galactose moiety of UDP-Gal (not shown) and a hydrogen bond with the oxygen atom of the phosphate group of the sugar nucleotide (Ramakrishnan and Qasba, 2001
). An earlier study showed that Trp314 is very important for catalysis (Aoki et al., 1990
), which is consistent with the structural evidence. The crystal structures of ß4Gal-T1and LA in complex with GlcNAc or Glc show that the carboxylate oxygens of Asp 318 and Asp 319 are hydrogen bonded to the monosaccharide (Ramakrishnan and Qasba, 2001
). The carboxylate oxygen of Asp318 forms hydrogen bonds with O3 and O4 of GlcNAc (or Glc) (Figure 7B) (Ramakrishnan and Qasba, 2001
). Furthermore, the interactions of N-butanoyl-GlcN with the Gal-T1 molecule in the N-butanoyl-GlcN-Gal-T1-LA complex are quite similar to the interactions of GlcNAc in the Gal-T1-LA-GlcNac complex (Ramakrishnan et al., 2001
).
In recent experiments using photoaffinity-labeled GlcNAc, Asp318 has been identified as the residue that binds the sugar acceptor (Hatanaka et al., 2001). Loss of activity associated with the substitution of Asp318 or Asp319 with either asparagine or alanine or glutamic acid is also consistent with the structural evidence (Ramakrishnan and Qasba, 2001
) and the mutational data of Zhang et al. (1999)
. However, the interpretation of the kinetic data presented by Zhang et al. (1999)
, which states that Asp318 does not directly affect GlcNAc binding, is not consistent with the structural and photoaffinity-labeling data. Mutation of Asp320 also affects the activity; in particular when it is substituted by asparagine. It then prefers Co2+ above Mn2+ as a metal ion. Substitution of Asp320 with glutamic acid, however, results in the retention of about 18% of the Mn2+-dependent activity of the wild type (Table IV). The mutation of Asp320 lowers the affinity for UDP-Gal and more strongly reduces the affinity for GlcNAc. This mutation, however, does not affect the metal binding to the high affinity site (K1) for Mn2+. In the crystal structure, Asp320 is not shown to make any contact with GlcNAc (Figure 7B). The carboxylate group of Asp320 is hydrogen-bonded with Tyr311 in both conformations I and II (Figures 7A and 7B). Substituting Tyr311 with glycine results in an increase in Km for GlcNAc by 1,000-fold and to a lesser extent (35-fold) the Km for UDP-Gal (Aoki et al., 1990
). The hydrogen bond between Asp320 and Tyr311 is likely to help stabilize the loop structure of the sequence region W312GWGGEDDD320, the region that is directly involved in GlcNAc binding and catalysis. Because the D320E mutant retains only 18% of the enzyme activity, the carboxylate group of the glutamic acid may still form a weak hydrogen bond with the hydroxyl group of Tyr311, stabilizing (albeit less efficiently) the loop structure.
Though our data are in agreement with the conclusion that the residues of the E317DDD320 motif do not constitute the high-affinity metal binding site (Zhang et al., 1999), they do however show that mutating Glu317 and Asp320 affect the Ca2+- or Co2+-dependent activation. In the absence of Mn2+, Ca2+ does not stimulate the enzyme activity of either the wild type or the mutants of Asp242 or Asp244. The wild-type protein and the mutants where the E317DDD320 site has not been altered can however be activated by a Ca2+ ion in the presence of low amounts of Mn2+ (Figure 5B). In contrast, Ca2+ inhibits the mutants of the E317DDD320 region, E317D and D320E at higher Mn2+ concentrations (Figure 5B). Because the ionic radius of Ca2+ is larger (0.99 Å) than that of Mn2+ (0.80 Å), Ca2+ may not fit in the mutated E317DDD320 region. Co2+, on the other hand, may be fitting better in the mutated region because it is seen to activate these mutants when low concentrations of Mn2+ are present (Figure 5A). One water molecule has been seen in the center of the loop structure that contains the sequence E317DDD320 hydrogen-bonded to Glu317 and the amino nitrogen of Asn356, which could be a potential second metal ion site (Ramakrishnan and Qasba, 2001
). Interestingly, there is some sequence identity between the sequence motif of W314GGEDDDIY322 of ß4Gal-T and the sequence motif W368GGAGKDIF376 of Serratia protease. The latter also adopts a loop conformation in the protein, and has been shown to coordinate a Ca2+ ion (Figure 8) (Baumann et al., 1995
). At present we do not have any structural data for metal binding to the W314GGEDDDIY322 region, but our data show that E317D and D320E or N behave differently toward Ca2+ and Co2+ activation compared to the wild type. Because the second metal binding site of ß4Gal-T1 is a low-affinity binding site, either high concentrations of Mn2+ or high concentrations of Ca2+ with low concentrations of Mn2+ may be required during crystallization to trap and localize the second metal ion. The mutants described herein are being investigated further for the localization of the second metal binding site of ß4Gal-T1.
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Materials and methods |
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Bacterial strains and growth conditions
Bacterial growth and transformation and plasmid isolation were carried out by standard procedures (Ausubel et al., 1987). Plasmid pEGT-d129, which encodes the catalytic domain (residues 130402) of bovine ß4Gal-T1, was used as the plasmid template for point mutations. Both wild-type and mutant ß4Gal-T1 were expressed in E. coli XL2-Blue ultracompetent cells. All the clones were grown in Luria-Bertani medium (LB) containing 50 µg/ml ampicillin at 37°C (Boeggeman et al., 1993
, 1995).
Site-directed mutagenesis
The protocol used for site-directed mutagenesis was from Clontech Laboratories (Transformer Site-Directed Mutagenesis Kit). All the mutants were constructed using the plasmid pEGT-d129, containing a Bam HI / Eco RI fragment of ß4Gal-T1, cloned into pET23a (Boeggeman et al., 1993). The transformation process included a selection primer, pNotApa, which was employed to introduce a unique restriction site in pEGT-d129 for further selection. The mutagenic primers were used to introduce the desired mutations. Amplifications of both the parental and mutated strands were accomplished in a repair-deficient E. coli strain, BMH71-18 muts. After digesting the parental plasmid with the selection-specific restriction enzyme (Not I), the mutant plasmids were amplified and cloned in E. coli XL2-Blue ultracompetent cells and BL21 (DE3)/pLysS competent cells (Boeggeman et al., 1993
, 1995). The mutagenic primers were from Genosys Biotechnologies or Recombinant DNA Laboratory (NCI, Frederick). The nucleotide sequences of the mutagenic primers used for mutagenesis are:
D242N: 5' pGAGGCCTTGAAGAACTATGACTACAACTGC-3'
D242A: 5' pGAGGCCTTGAAGGCCTATGACTACAACTGC-3'
D242E: 5' pGAGGCCTTGAAGGAATATGACTACAACTGC-3'
D244N: 5' pGAGGCCTTGAAGGACTATAACTACAACTGC-3'
D244A: 5' pGAGGCCTTGAAGGACTATGCCTACAACTGC-3'
D244E: 5' pGAGGCCTTGAAGGACTATGAATACAACTGC-3'
D252N: 5' pGCTTTGTGTTTAGCAATGTCGACCTCATCCCA-3'
D252A: 5' pGTGTTTAGCGCTGTCGACCTCATCCCAATG-3'
D252E: 5' pGTGTTTAGCGAAGTCGACCTCATCCCAATG-3'
D254N: 5' pGTGTTTAGCGATGTGAACCTCATCCCAATGA-3'
D254A: 5' pGTGTTTAGCGATGTCGCCCTCATCCCAATGA-3'
D254E: 5' pGTGTTTAGCGATGTCGAACTCATCCCAATGA-3'
E317Q: 5' pGGGGCTGGGGAGGTCAAGATGATGACATTTATAAC-3'
E317A: 5' pGGCTGGGGAGGTGCAGATGATGACATTTAT-3'
E317D: 5' pGGCTGGGGAGGTGACGATGATGACATTTAT-3'
D318N: 5' pGGGGCTGGGGAGGTGAAAATGATGACATTTATAAC-3'
D318A: 5' pGGCTGGGGAGGTGAAGCTGATGACATTTAT-3'
D318E: 5' pGGCTGGGGAGGTGAAGAAGATGACATTTAT-3'
D319N: 5' pGGGGAGGTGAAGATAATGACATTTATAACAGATTAGC-3'
D319A: 5' pGGCTGGGGAGGTGAAGATGCTGACATTTAT-3'
D319E: 5' pGGCTGGGGAGGTGAAGATGAAGACATTTAT-3'
D320N: 5' pGGGGAGGTGAAGATGATAACATTTATAACAGATTAGC-3'
D320E: 5' pGGGGAGGTGAAGATGATGAAATTTATAACAGATTAGC-3'
M344A: 5' pGTGATCGGGAAGACGCGCGCGATCCGCCACTCGAGAGAGAAGAAA-3'
M344Q: 5' pGTGATCGGGAAGACGCGCCAGATCCGCCACTCGAGAGACAAGAAA-3'
H347E: 5' pGTGATCGGGAAGACGCGCATGATCCGCGAATCGAGAGACAAGAAA-3'
H347D: 5' pGTGATCGGGAAGACGCGCATGATCCGCGACTCGAGAGACAAGAAA-3'
H347N: 5' pGTGATCGGGAAGACGCGCATGATCCGCAACTCGAGAGACAAGAAA-3'
H347Q: 5' pGTGATCGGGAAGACGCGCATGATCCGCCAGTCGAGAGACAAGAAA-3'
The mutated residues are shown in italics. The potential mutants were selected after observing changes in the restriction enzyme patterns. Mutations were confirmed by DNA sequencing using Sequenase version 2.0 (USB, Cleveland, OH).
Preparation and purification of wild-type and mutant proteins
The E. coli XL2-Blue ultracompetent and BL21 (DE3)/pLysS competent cells were transformed with the pET vector derivatives per manufacturers protocols. The transformed cells were grown overnight with shaking at 37°C in an LB broth containing 50 µg ml1 ampicillin. The overnight cultures were induced with 0.4 mM ispropylthiogalactoside (IPTG) for 4 h. After IPTG induction, the inclusion bodies were isolated from the bacterial pellet essentially as described (Boeggeman et al., 1993). After washing four times with a suspension buffer containing 25% (w/v) sucrose, the inclusion bodies were washed with 1x phosphate buffered saline buffer. From a liter of induced bacterial culture, one generally gets ~80100 mg of purified inclusion bodies. Our laboratory has developed a protocol to generate active recombinant ß4Gal-T1 from these inclusion bodies using oxido-shuffling agents during renaturation (Boeggeman et al., 1993
). On dilution with guanidine-HCl and subsequent dialysis, a portion of the folded protein was found to precipitate in the form of aggregates. To increase the protein yields, we modified this protocol as follows: 100 mg of inclusion bodies were dissolved in 10 ml of 5 M guanidine-HCl containing 0.3 M sodium sulfite. One milliliter of a 50 mM disodium 2-nitro-5-thiosulfobenzoate solution is added to sulfonate the free thiols in the proteins. Completion of sulfonation was judged by a color change from deep orange to pale yellow. The sulfonated protein was diluted 10-fold in water, precipitated, collected by centrifugation at 10,000 x g, and washed four times with water to remove any remaining sulfonating agent. The protein was redissolved in 5 M guanidine-HCl, to a protein concentration of 1 mg/ml, with OD275 of 1.92.0. The protein solution was diluted 10-fold in a solution containing 100 µg/ml ß4Gal-T1, 50 mM TrisHCl (pH 8.0), 5 mM ethylenediamine tetra-acetic acid, 0.5M guanidine-HCl, 4 mM cysteamine, and 2 mM cystamine. The protein was renatured for 48 h at 4°C, dialyzed, and concentrated using ultrafiltration membranes (Amicon, Beverly, MA). The advantage of this method is that the precipitated protein can be resulfonated to generate more of active ß4Gal-T1. Using this procedure, the yield was 35 mg of pure and active ß4Gal-T1 from 100 mg sulfonated protein per liter of folding solution. The precast Novex gels were used for SDSPAGE analysis, and the protein bands visualized with Coomassie blue. Protein concentrations were measured with the Bio-Rad protein dye reagent with bovine serum albumin as the standard.
ß4Gal-T1 enzyme assays and kinetic analysis
The in vitro assay for ß4Gal-T1 enzyme activity was performed as described (Boeggeman et al., 1995). The activities were measured using 3H-labeled-UDP-Gal and the sugar acceptor GlcNAc. Typically, a 100-µl incubation mixture contained 25 mM GlcNAc, 5 mM MnCl2, 0.05 mM UDP-Gal, 5 mM TrisHCl (pH 8.0), 0.5% Triton X-100, 1020 ng of ß4Gal-T1, and 0.5 µCi 3H-UDP-Gal. A reaction without GlcNAc was used as a control. The incubation was carried out at 30°C for 15 min and terminated by the addition of 200 µl cold water. The incubation mixture was added to a column containing 0.5 ml bed volume AG1-X8 cation resin and was washed with cold water. The flow-through was diluted in 20 ml Biosafe scintillation fluid and the radioactivity measured in a Beckman counter. The mutated proteins showed reduced but detectable enzymatic activities as compared to the wild-type recombinant ß4Gal-T1.
The kinetic parameters for UDP-Gal and GlcNAc were determined using double substrate kinetics at a fixed Mn2+ concentration of 10 mM, with concentrations of UDP-Gal varying between 0.005 and 2 mM and at fixed GlcNAc concentration of 150 mM. Data were analyzed for a two-substrate system by fitting to an equation for sequential symmetrical initial velocity pattern, Equation 1 (Zhang et al., 1999), (ordered or random equilibrium mechanism); or to an equation for asymmetric initial velocity pattern, Equation 2 (Zhang et al., 1999
), for a double-displacement or ping-pong mechanism.
(1) = (Vmax* [A] * [D]) / (Kid * Kmd) + (Kmd * [A]) + (Kma * [D]) +
([A] * [D])
(2) = (Vmax * [A] * [D]) / (Kma * [D]) + (Kmd * [A]) + ([A] * [D])
In the equations, [D] represents the concentration of the donor; [A] the concentration of the acceptor; Kma and Kmd, the true Km for the acceptor and the donor, respectively; and Kid the dissociation constant for the donor. The Enzfitter (Biosoft), a nonlinear curve-fitting program for Windows was used to obtain the kinetic parameters from the fitted curves using Equations 1 and 2.
For determining metal binding constants the rate of N-acetyllactosamine synthesis was measured at various concentrations of metal ions at fixed GlcNAc (20 mM) and UDP-Gal concentrations (0.2 mM). Mn2+ and Co2+ concentrations were varied between 0 and 5 mM. The data were fitted to Equation 3 (Zhang et al., 1999), describing two-metal binding sites, or to Equation 4 (Zhang et al., 1999
), describing a single-metal binding site.
(3) = [M] * [(V1 * K2) + (V2 * [M])] / [(K1 * K2) + (K2 * [M] +[M]2)]
(4) = (V * [M]) / (Kd +[M])
The metal binding to site 1 with high affinity (K1) is associated with low velocity (V1) and the binding to the low-affinity site 2 with an apparent Kd of K2 is associated with high velocity (V2). [M] is the concentration of either Mn2+ or Co2+.
Binding of recombinant proteins to UDP-affinity column
The binding to the UDP-agarose column was carried out at 4°C. Columns containing 1 ml bed volume of UDP-agarose were preequilibrated with a 25 mM cacodylic buffer, pH 7.6, containing variable amounts of MnCl2 (25, 5, or 1 mM). The renatured protein solutions (0.5 mg) were adjusted to the preequilibration buffer conditions and applied to the columns. After loading of the samples, the pass-through was recycled again. The pass-through was analyzed for the unbound protein (U). The columns were washed 3x with 1-ml portions of equilibration buffer (W). The elution buffer consisted of 25 mM cacodylic buffer, pH 7.6, containing 25 mM ethylenediamine tetra-acetic acid and 1 M NaCl. For elution of the proteins, 0.5-ml fractions (E) were collected from the columns, and aliquots were analyzed by SDSPAGE.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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