Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA
Received on May 30, 2001; revised on August 17, 2001; accepted on August 29, 2001.
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Abstract |
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Key words: cysteine residues/disulfide bonds/HA biosynthesis/mutagenesis/synthase
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Introduction |
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We previously purified the recombinant spHAS and seHAS enzymes, expressed in Escherichia coli (Tlapak-Simmons et al., 1999a) and characterized both enzymes with respect to their kinetic constants (Tlapak-Simmons et al., 1999b
), functional size (Tlapak-Simmons et al., 1998
), and their requirement of a phospholipid, particularly cardiolipin, for enzyme activity (Tlapak-Simmons et al., 1999a
). Although the streptococcal HASs are relatively small at <49 kDa, they mediate at least six discrete functions: the ability to bind two different sugar nucleotide precursors, to catalyze two distinct glycosyltransferase reactions, to bind the HA acceptor polymer, and to translocate the growing HA chain through the enzyme and the cell membrane.
We recently found that spHAS and seHAS are sensitive to sulfhydryl reagents, such as iodoacetamide or NEM, which partially inhibit enzyme activity (Kumari et al., unpublished data). This inhibition implicates the involvement of one or more Cys residues in at least one of these six distinct activities of HAS required for HA biosynthesis. There are six Cys residues in spHAS, four of which are conserved perfectly in seHAS and suHAS (Figure 1); both of these latter enzymes have only four Cys residues (Kumari and Weigel, 1997; Ward et al., 2001
). These four Cys residues in turn are generally conserved among the three vertebrate HAS isoenzymes (Weigel et al., 1997
). No one has yet addressed the possibilities that one or more of these conserved Cys residues within the Class I HAS family may be critical for enzyme activity or may participate in the formation of disulfide bonds.
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Results |
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Therefore, to normalize for the level of HAS protein expression, we developed a sensitive and quantitative western blotbased assay (Heldermon et al., 2001). Because all of our HAS constructs contain a C-terminal His6 tag, which is efficiently recognized by a commercial anti-His5 monoclonal antibody, we used this antibody after biotinylation as the primary antibody for analysis of western blots followed by incubation with 125I-streptavidin as the secondary reagent. Unlike standard western analysis, this detection protocol provides greater sensitivity as well as the ability to quantitate HAS protein over a much broader concentration range. The normalizations for HAS protein expression were performed relative to known amounts of purified spHAS-His6 included in each analysis as internal standards. Based on the normalized results, it was clear that spHAS(C225S) was expressed at the lowest level relative to any of the other mutants,
66% of wild type (Table I). The protein expression levels for the majority of single Cys-mutants were not significantly different than wild type, although the spHAS(C124S) and spHAS(C261A) variants may have been elevated by
35% (P
0.05). Interestingly, most of the multiple Cys-mutants as well as the Cys-null mutant were expressed at three- to fivefold higher levels than the wild-type enzyme. These differences in relative expression of these spHAS variants were consistent in multiple experiments, with independent cell growth and enzyme induction, indicating that several of the Cys residues in spHAS, particularly the conserved Cys at position 225, may influence the initial folding and stability of the enzyme.
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The KUDP-GlcUA values for all of the single Cys-mutants and the two double Cys-to-Ala mutants were within 50% of wild type. The remaining multiple Cys-to-Ala mutants exhibited KUDP-GlcUA values that were two to three times that of wild type. Although these multiple Cys-mutations do alter the activity of the enzyme by decreasing the efficiency of utilizing UDP-GlcUA, they do not do so in a large way. Furthermore, the relatively modest difference in activity between the Cys-null mutant and wild type spHAS clearly shows that cysteine residues are not absolutely necessary for HA synthesis, either catalytically or structurally.
Inhibition of spHAS activity by NEM
Our initial finding that the wild-type streptococcal HAS enzymes were inhibited by NEM (Kumari et al., unpublished data) was the impetus for investigating the role of Cys residues in the enzyme. NEM treatment of membranes from a panel of multiple Cys-mutants showed that this inhibition was no longer present in spHASCysnull or the mutant with only Cys225 intact, whereas NEM sensitivity remained in the other multiple Cys-mutants (Figure 4). These results indicate that the inhibition of the wild-type enzyme by NEM or other sulfhydryl-reactive agents is most likely due to modification of the Cys residues alone, rather than the loss of the S-H group. The lack of inhibition of the single Cys-containing mutant demonstrates that Cys225 is either predominantly inaccessible to modification by NEM due to its position in the enzyme or that this particular cysteine residue is not involved in the inhibition response of the enzyme when modified by NEM.
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Discussion |
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Finally, despite the high degree of conservation of four of the six Cys residues within the larger HAS family and their complete conservation among the three streptococcal HAS enzymes (Figure 1), the spHASCysnull mutant was completely insensitive to inhibition by NEM or other sulfhydryl-reactive reagents. Cys225 is the best candidate Cys residue to explain the NEM sensitivity of spHAS, seHAS, and probably the other HAS enzymes as well, because this Cys residue is the only one that is absolutely conserved in the Class I HAS family (Figure 1). A surprising result, therefore, was that the quintuple-Cys mutant of spHAS containing only Cys225 was not inhibited by NEM or other -SH modifying agents. Although spHAS is functional even with Cys225 replaced by Ser or Ala, ongoing experiments (Kumari et al., unpublished data) indicate that this highly conserved residue may be near or within one of the sugar nucleotide binding sites and may participate in HA biosynthesis in an as-yet-undefined way.
Based on all of the above results we conclude that the role of the Cys residues in the structure or catalysis of the streptococcal HAS proteins must be subtle, because replacement of these residues with Ala does not substantially inhibit the enzymes activity or, therefore, presumably its structure. Because the Cys-null mutant is active, this subtle structural role cannot be essential unless there is a substantial difference in behavior of this mutant enzyme in live cells compared to isolated membranes or the detergent-solubilized, purified protein. Studies are in progress to test this possibility.
One possible functional role for the Cys residues in spHAS, which would not be revealed by the present analysis, is in the control of HA product size. It is possible for the HAS enzymes in general that distinct mechanisms control HA chain product length, that is, the size distribution of the HA chains produced, and the overall HA biosynthesis capacity (Abatangelo and Weigel, 2000). Another important consideration in evaluating the importance of Cys residues in spHAS (and the other Class I HAS family members in general) may be their involvement in HA translocation. The mechanism by which these enzymes are able to hold onto the growing HA chain while they continuously extrude the polysaccharide through the bacterial cell or plasma membrane is still unknown. We refer to this extrusion process as a translocation, because the HA is not completely transferred across the membrane and released as would occur in a typical transport process. The synthesis and extracellular accumulation by some bacteria of polysaccharides, such as polysialic acid, often requires multiple factors and proteins encoded by very complex multigene operons (Moxon and Kroll, 1990
; Bliss and Silver, 1996
). In contrast, all of the genetic and biochemical evidence to date (reviewed in Weigel, 1998
) demonstrate that the streptococcal enzymes are able to initiate HA chain formation and then rapid extension of the HA chain in the absence of any primer or other proteins. Other than the two sugar nucleotide substrates and Mg2+, the purified spHAS or seHAS enzymes only require a phospholipid (Tlapak-Simmons et al., 1999a
) to produce high-molecular-weight HA (>106 Da). In particular, cardiolipin dramatically stimulates the specific activity of detergent solubilized or purified spHAS and seHAS. The size distribution of HA products is very similar for enzyme in isolated membranes or after solubilization with dodecylmaltoside and affinity purification (data not shown). Therefore, the presence of a natural intact phospholipid bilayer and membrane does not affect the ability of the HAS enzymes to synthesize HA. Presently we do not have a suitable assay to evaluate the ability of the wild-type or Cys-mutant enzymes to translocate HA.
The creation of a spHASCysnull mutant that retains enzymatic activity should enable a more in-depth analysis of the tertiary structure of spHAS and possible conformation changes during substrate binding, catalysis, or HA translocation. To understand these processes, it is necessary to determine the molecular proximity and interactions of various domains within the protein in a more defined way. For example, as reported for the Lac permease (Frillingos et al., 1998), Cys-scanning mutants of spHAS containing a single unique Cys residue at a desired position may enable one to perform electron paramagnetic resonance studies by modifying this Cys residue with a suitable probe to determine the proximity of that residue to another region of the molecule (Voss et al., 1997
). Similarly, chemical modification of a single unique Cys residue with a fluorescent probe may enable analysis of the localized environment within different regions of the protein (Jung et al., 1994
). Finally, Cys-scanning or site-specific mutagenesis followed by assessment of possible disulfide bond formation (Wang and Kaback, 1999
) could help establish interacting or proximal domains within HAS. These approaches, based on the present finding that spHASCysnull is active, hold promise for elucidating the structure and function of spHAS and furthering our understanding of the synthesis of large glycosidic polymers.
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Materials and methods |
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Mutagenesis of Cys residues
Single mutants were generated by the Altered Sites Mutagenesis or Quick Change Mutagenesis protocols using primers (Table III) designed to change the Cys residues at positions 124, 225, 261, 280, 366, or 402 of spHAS containing His6 at the C-terminus (Tlapak-Simmons et al., 1999a). After generating and confirming the entire sequence of each spHAS(Cys-to-Ser) mutant produced in the Altered Sites vector, internal restriction sites within the HAS ORF were used to transfer mutated regions to the spHAS insert in pKK223 (this vector carrying HAS is designated pKK3K). Cys-to-Ala mutants of spHAS were generated directly in the pKK3K vector using the Quick Change mutagenesis method. Site-directed mutagenesis was used to generate the C124,C402A double mutant, and then C366A was added by restriction fragment exchange to generate a triple mutant. Site-directed mutagenesis was also used to create the double mutant spHAS(C261A,C280A). The mutants containing five or six mutated Cys residues were generated by utilizing restriction sites to combine fragments of spHAS containing different mutations. For example, AvrII and MfeI were used to combine the spHAS(C124A,C366A,C402A) triple Cys-mutant and the spHAS(C261A,C280A) double mutant to create spHAS with only Cys225 intact. Finally, BglII and AvrII were used to splice spHAS(C225A) into the latter quintuple Cys-mutant to generate the Cys-null clone, designated spHASCysnull. All Cys-to-Ala/Ser mutants were confirmed over the full ORF by automated DNA sequencing.
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Enzymatic analysis of HAS mutants
E. coli SURE cells, previously transformed with plasmids containing wild-type or mutant spHAS genes, were grown to an A600 of 1.2 and induced with 1 mM isopropyl thio-ß-D-galactoside for 3 h. Cells were harvested, and membranes were prepared as described previously (Tlapak-Simmons et al., 1999a
). The activities of mutant spHAS variants were assessed by measuring their Vmax and Km values in isolated membranes, normalized as described above for the amount of enzyme expressed. The Km values were determined using a descending paper chromatography assay (Tlapak-Simmons et al., 1999b
), holding one UDP-sugar substrate constant and varying the other from 0.01 to 4 mM. Data were analyzed by linear regression using Haynes-Wolf plots for UDP-GlcUA or Hill plots for UDP-GlcNAc.
Inhibition of spHAS activity by NEM
Membrane preparations from wild-type and various spHAS mutants (i.e., C124,402A, C261,280A, C124,366,402A, C124,261,280,366,402A, and the Cys-null mutant) were incubated in 50 mM sodium, potassium phosphate, pH 7.0, 75 mM NaCl and 10% (v/v) glycerol with or without 20 mM NEM for 90 min on ice. The ability of the membrane samples to synthesize HA was then assessed by adding the following to the final concentrations indicated: 1 mM UDP-GlcUA, 1 mM UDP-GlcNAc, 0.68 µM UDP-[14C]GlcUA in 25 mM sodium/potassium phosphate, pH 7, 75 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 0.1 mM ethylene glycol bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 15% glycerol. Reactions were shaken for 1 h at 30°C in a Taitec E-36 micromixer, stopped by the addition of SDS to 2% (w/v), and spotted onto No. 3MM Whatman paper for descending paper chromatography overnight using 1 mM ammonium acetate pH 5.5:ethanol (7:13). [14C]GlcUA incorporation into high-molecular-weight HA was assessed by liquid scintillation spectroscopy to determine the radioactivity remaining at the origin. Confirmation that the latter material is authentic HA was obtained by showing its complete loss after treatment with streptomyces hyaluronidase.
Assessment of disulfide bond formation
Wild-type spHAS-His6 was bound to a Ni2+ chelate column (Qiagen) and washed as previously described (Tlapak-Simmons et al., 1999a). While still bound to the resin, the enzyme was incubated with biotin-PEO-maleimide (10 mg/ml) in the presence or absence of 6 M guanidinium-HCl for 2 h at 4°C. The column was washed, and spHAS-His6 was eluted with distilled water containing 0.5% (v/v) trifluroacetic acid and 0.02% (w/v) dodecylmaltoside. To assess the degree of modification of Cys residues, samples containing purified spHAS were analyzed by MALDI-TOF MS using a Voyager Elite mass spectrometer (Applied Biosystems, Framingham, MA), which was equipped with a N2 laser (337 nm), located in the NSF EPSCoR Oklahoma Laser Mass Spectrometry Facility. A 1-µl aliquot of sample was spotted to a sample plate followed by 1 µl of matrix and allowed to air-dry. The matrix used was a 20 mg/ml solution of 2,4,6-trihydroxyacetophenone in 50% acetonitrile containing 0.1% trifluoroacetic acid and 0.05% (w/v) dodecylmaltoside. Samples were analyzed in the linear, positive ion mode using a delayed extraction of 300 ns and a grid voltage of 87.8%, and they were subject to a 25 kV accelerating voltage. External calibrations were performed routinely using horse apomyoglobin and bovine serum albumin (16,951 and 66,430 Da, respectively). Data were routinely processed using the 19-point Savitsky-Golay smoothing option included in the software provided by the manufacturer.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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Bliss, J.M. and Silver, R.P. (1996) Coating the surface: a model for expression of capsular polysialic acid in Escherichia coli K1. Mol. Microbiol., 21, 221231.[ISI][Medline]
DeAngelis, P.L. (1999) Hyaluronan synthases: fascinating glycosyltransferases from vertebrates, bacterial pathogens, and algal viruses. Cell. Mol. Life Sci., 56, 670682.[ISI]
DeAngelis, P.L., Papaconstantinou, J., and Weigel, P.H. (1993a) Isolation of a Streptococcus pyogenes gene locus that directs hyaluronan biosynthesis in acapsular mutants and in heterologous bacteria. J. Biol. Chem., 268, 1456814571.
DeAngelis, P.L., Papaconstantinou, J., and Weigel, P.H. (1993b) Molecular cloning, identification, and sequence of the hyaluronan synthase gene from Group A Streptococcus pyogenes. J. Biol. Chem., 268, 1918119184.
DeAngelis, P.L., Jing, W., Drake, R.R., and Achyuthan, A.M. (1998) Identification and molecular cloning of a unique hyaluronan synthase from Pasteurella multocida. J. Biol. Chem., 273, 84548458.
DeAngelis, P.L., Jing, W., Graves, M.V., Burbank, D.E., and Van Etten, J.L. (1997) Hyaluronan synthase of chlorella virus PBCV-1. Science, 278, 18001803.
Evered, D. and Whelan, J. (eds) (1989) The biology of hyaluronan. Ciba Fnd. Symp., 143, 1288.
Fenderson, B.A., Stamenkovic, I., and Aruffo, A. (1993) Localization of hyaluronan in mouse embryos during implantation, gastrulation and organogenesis. Differentiation, 54, 8598.[ISI][Medline]
Frillingos, S., Sahin-Toh, M., Wu, J., and Kaback, H.R. (1998) Cys-scanning mutagenesis: a novel approach to structure-function relationships in polytopic membrane proteins. FASEB J., 12, 12811299.
Heldermon, C.D., DeAngelis, P.L., and Weigel P.H. (2001) Topological organization of the hyaluronan synthase from Streptococcus pyogenes. J. Biol. Chem., 276, 20372046.
Hill, A.V. (1913) The combinations of haemoglobin with oxygen and with carbon monoxide. Biochem. J., 7, 471480.
Itano, N. and Kimata, K. (1996a) Expression cloning and molecular characterization of HAS protein, a eukaryotic hyaluronan synthase. J. Biol. Chem., 271, 98759878.
Itano, N. and Kimata, K. (1996b) Molecular cloning of human hyaluronan synthase. Biochem. Biophys. Res. Commun., 222, 816820.[ISI][Medline]
Jung, K., Jung, H., and Kaback, H.R. (1994) Dynamics of lactose permease of Escherichia coli determined by site-directed fluorescence labeling. Biochemistry, 33, 39803985.[ISI][Medline]
Knudson, C.B. and Knudson, W. (1993) Hyaluronan-binding proteins in development, tissue homeostasis, and disease. FASEB J., 7, 12331241.
Kumari, K. and Weigel, P.H. (1997) Molecular cloning, expression, and characterization of the authentic hyaluronan synthase from Group C Streptococcus equisimilis. J. Biol. Chem., 272, 3253932546.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685.[ISI][Medline]
Laurent, T.C. and Fraser, J.R.E. (1992) Hyaluronan. FASEB J., 6, 23972404.
Lin, Y., Mahan, K., Lathrop, W.F., Myles, D.G., and Primakoff, P. (1994) A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg. J. Cell Biol., 125, 11571163.[Abstract]
Moxon, E.R. and Kroll, J.S. (1990) The role of bacterial polysaccharide capsules as virulence factors. Curr. Top. Microbiol. Immunol., 6, 6585.
Shyjan, A.M., Heldin, P., Butcher, E.C., Yoshino, T., and Briskin, M.J. (1996) Functional cloning of the cDNA for a human hyaluronan synthase. J. Biol. Chem., 271, 2339523399.
Spicer, A.P., Augustine, M.L., and McDonald, J.A. (1996) Molecular cloning and characterization of a putative mouse hyaluronan synthase. J. Biol. Chem., 271, 23400-23406.
Tlapak-Simmons, V.L., Baggenstoss, B.A., Clyne, T., and Weigel, P.H. (1999a) Purification and lipid dependence of the recombinant hyaluronan synthases from Streptococcus pyogenes and Streptococcus equisimilis. J. Biol. Chem., 274, 42394245.
Tlapak-Simmons, V.L., Baggenstoss, B.A., Kumari, K., Heldermon, C., and Weigel, P.H. (1999b) Kinetic characterization of the recombinant hyaluronan synthases from Streptococcus pyogenes and Streptococcus equisimilis. J. Biol. Chem., 274, 42464253.
Tlapak-Simmons, V.L., Kempner, E.S., Baggenstoss, B.A., and Weigel, P.H. (1998) The active streptococcal hyaluronan synthases (HASs) contain a single HAS monomer and multiple cardiolipin molecules. J. Biol. Chem., 273, 2610026109.
Toole, B.P. (1997) Hyaluronan in morphogenesis. J. Intern. Med., 242, 3540.[ISI][Medline]
Towbin, H., Steahelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. Sci. USA, 76, 43504354.[Abstract]
Voss, J., Hubbell, W.L., Hernandez-Borrell, J., and Kaback, H.R. (1997) Site-directed spin-labeling of transmembrane domain VII and the 4B1 antibody epitope in the lactose permease of Escherichia coli. Biochemistry, 36, 1506115066.
Wang, Q. and Kaback, H.R. (1999) Location of helix III in the lactose permease of Escherichia coli as determined by site-directed cross-linking. Biochemistry, 38, 1677716782.[ISI][Medline]
Ward, P.N., Field, T.R., Ditcham, W.G., Maguin, E., and Leigh, J.A. (2001) Identification and disruption of two discrete loci encoding hyaluronic acid capsule biosynthesis genes hasA, hasB and hasC in Streptococcus uberis. Infect. Immun., 69, 392399.
Watanabe, K. and Yamaguchi, Y. (1996) Molecular identification of a putative human hyaluronan synthase. J. Biol. Chem., 271, 2294522948.
Weigel, P.H. (1998) Bacterial hyaluronan synthases. In Hascall, V.C., and Yanagishita, M., (eds), Science of hyaluronan today. Available online at: www.GlycoForum.gr.ip.
Weigel, P.H., Hascall, V.C., and Tammi, M. (1997) Hyaluronan synthases. J. Biol. Chem., 272, 1399714000.
Wessels, M.R., Goldberg, J.B., Moses, A.E., and Dicesare, T.J. (1994) Effects on virulence of mutations in a locus essential for hyaluronic acid capsule expression in Group A streptococci. Infect. Immun., 62, 433441.[Abstract]
Wessels, M.R., Moses, A.E., Goldberg, J.B., and Dicesare T.J. (1991) Hyaluronic acid capsule is a virulence factor for mucoid Group A streptococci. Proc. Natl Acad. Sci. USA, 88, 83178321.[Abstract]
Whitnack, E., Bisno, A.L., and Beachey, E.H. (1981) Hyaluronate capsule prevents attachment of Group A streptococci to mouse peritoneal macrophages. Infect. Immun., 31, 985991.[ISI][Medline]