C-terminal Position Research Articles

Constrained analogs of methionine: asymmetric synthesis of χ-angle restricted methionine derivatives through [3.1.0]-γ-lactone cyclopropane ring opening

Regiospecific and stereoselective ring opening of a [3.1.0]-γ-lactone intermediate by sulfur-containing nucleophiles has been developed. The reaction product was confirmed by NMR techniques and chemical equilibrium processes. Using this approach, all four possible diastereomers of χ-angle restricted methionine surrogates have been synthesized by electrophilic azidation and reduction to the amino group. These constrained analogs can be used in the preparation of peptidomimetics of sequences containing methionine in the C-terminal position.

Hydrolysis of sequenced beta-casein peptides provides new insight into peptidase activity from thermophilic lactic acid bacteria and highlights intrinsic resistance of phosphopeptides.

The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified beta-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the beta-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the beta-casein hydrolysate as the substrate at pH 5.5 and 24 degrees C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing beta-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the beta-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species.

Expression and purification of histidine-tagged proteins from the gram-positive Streptococcus gordonii SPEX system.

Streptococcus gordonii (S. gordonii) has been used as a gram-positive bacterial expression vector for secreted or surface-anchored recombinant proteins. Fusion of the gram-positive bacterial N-terminal signal sequence to the target protein is all that is required for efficient export. This system is termed SPEX for Surface Protein EXpression and has been used to express proteins for a variety of uses. In this study, the SPEX system has been further developed by the construction of vectors that express polyhistidine-tagged fusion proteins. SPEX vectors were constructed with an N-terminal or C-terminal histidine tag. The C-repeat region (CRR) from Streptococcus pyogenes M6 protein and the Staphylococcus aureus nuclease A (NucA) enzyme were tested for expression. The fusion proteins were purified using metal affinity chromatography (MAC). Results show that the fusion proteins were expressed and secreted from S. gordonii with the His tag at either the N- or C-terminal position and could be purified using MAC. The M6 fusions retained immunoreactivity after expression and purification as determined by immunoblots and ELISA analyses. In addition, NucA fusions retained functional activity after MAC purification. The M6-His and NucA-His fusions were purified approximately 15- and 10-fold respectively with approximately 30% recovery of protein using MAC. This study shows that the polyhistidine tag in either the N- or C-terminal position is a viable way to purify secreted heterologous proteins from the supernatant of recombinant S. gordonii cultures. This study further illustrates the value of the SPEX system for secreted expression and purification of proteins.

Spacer length dependence on the efficiency of dimeric anionic peptides in gene transfer by glycosylated polylysine/plasmid complexes.

Amphiphilic anionic peptides have been used to enhance the efficiency of transfection by helping plasmids to escape from endosomes to the cytosol. It has been shown that efficiency of an eicosamers containing five glutamyl residues (E5), can be considerably enhanced either by transforming it into a dimer or by adding a tripeptide WYG in a C-terminal position (E5WYG). The dimerization of the peptide E5WYG leads to a more efficient tool when the dimerization device includes the tripeptide WYG unit and a longer spacer arm made of Gly-betaAla-betaAla residues, but to a 10-fold less efficient tool when the dimerization device includes a shorter spacer, a glycyl residue. Both dimers are taken up by the cells to a similar extent. Both dimers seem to be surrounded similarly as far as the environmental pH is concerned. In contrast, we found a correlation between the propensity of the peptides to adopt a helical structure at neutral pH and the gene transfer efficiency.

Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct

Hexahistidine tag (His-tag) is the most widely used tag for affinity purification of recombinant proteins for their structural and functional analysis. In the present study, single chain Fv (scFv) constructs were engineered form the monoclonal antibody (MAb) CC49 which is among the most extensively studied MAb for cancer therapy. For achieving efficient purification of scFvs by immobilized metal–ion affinity chromatography (IMAC), a His-tag was placed either at the C-terminal (scFv-His 6) or N-terminal (His 6-scFv) of the coding sequence. Solid-phase radioimmunoassay for scFv-His 6 showed only 20–25% binding whereas both His 6-scFv and scFv (no His-tag) showed 60–65% binding. Surface plasmon resonance studies by BIAcore revealed the binding affinity constant ( K A) for His 6-scFv and scFv as 1.19×10 6 M −1 and 3.27×10 6 M −1, respectively. No K A value could be calculated for scFv-His 6 due to poor binding kinetics ( k on and k off). Comparative homology modeling for scFv and scFv-His 6 showed that the C-terminal position of the His-tag partially covered the antigen-binding site of the protein. The study demonstrates that the C-terminal position of His-tag on the CC49 scFv adversely affects the binding properties of the construct. The results emphasize the importance of functional characterization of recombinant proteins expressed with purification tags.

Alternative splicing of the NMDAR1 glutamate receptor subunit in human temporal lobe epilepsy

It has been demonstrated in animal models that chronic epilepsy is associated with increased excitability which may result from abnormal glutamatergic transmission involving altered properties of N-methyl- d-aspartate (NMDA) receptors. We have investigated whether human temporal lobe epilepsy is associated with changes in the NMDA receptor at the molecular level by assessing the relative expression of mRNAs of the different splice variants at the N-terminal (exon 5) and C-terminal (exon 21) position for the NMDAR1 subunit. Specimens of hippocampus and temporal lobe cortex from patients with refractory epilepsy were obtained during neurosurgical operations and analyzed by means of the reverse transcription reaction followed by polymerase chain reaction. Non-epileptic control specimens obtained at autopsy exhibited a relatively high level in expression of exon 5-lacking (hippocampus: 0.87; cortex: 0.81) and exon 21-containing (hippocampus: 0.95; cortex: 0.93) transcripts. The ratio for these alternatively spliced transcripts was not significantly changed in epileptic hippocampal and cortical tissues relative to the corresponding non-epileptic samples. These results did not support a potential role for NMDAR1 splice variants in the pathophysiology of epilepsy.

FMRFamides exert a unique modulation of rodent pancreatic polypeptide sensitive neuropeptide Y (NPY) receptors

FMRFamide and related peptides (RFamides) were found to inhibit the association binding of iodinated human pancreatic polypeptide ([125I]hPP) to Y5-like neuropeptide Y (NPY) receptor in rodent tissues. An allosteric regulation of the activity of the rodent kidney PP-sensitive neuropeptide Y (NPY) receptor by RFamides was indicated by potency decrease with particle concentration in the inhibition of the association binding of 125I-labeled human pancreatic polypeptide (hPP) by RFamides at rabbit kidney membranes. The competition by C-terminal hexapeptide of hPP (LTRPRY.NH2) did not show such affinity change. The steady-state binding of hPP showed little sensitivity to any of the RFamides tested. The Y1-selective binding of [125I][Leu31,Pro34]hPYY (at 2 nM hPP) was much less sensitive to RFamides than the binding of [125I]hPP, albeit with some differences across tissue or cell types. The binding of Y2-selective agonist 125I-labeled human peptide YY (3-36) was quite insensitive to RFamides. The presence of a unique component in the inhibition of hPP binding by RFamides was further indicated by a degree of antagonism with phospholipase C inhibitor U-73122, and by an only limited cooperation with a N5-amiloride compound, and with alkylator chloroethylclonidine. Change of the chirality of individual residues in the FMRFamide molecule produced a significant reduction of inhibitory potency only with D-Phe in the C-terminal position. Substitution of the (C-3) L-Met by L-Leu greatly increased the inhibitory potency of RFamides relative to otherwise identical congeners. RFamides could act both as ligands of membrane neighbors of the PP receptor, and as competitors of Y5-like NPY receptor epitopes that accommodate the C-terminal aspects of agonist peptides.

Activation of protein kinase C induces internalization of the GABA(C) receptors expressed in Xenopus oocytes.

In a previous study, we showed that the protein kinase C (PKC) activator 4beta-phorbol 12-myristate 13-acetate (PMA) inhibited the gamma-aminobutyric acid (GABA)-gated currents in Xenopus oocytes expressing human rho1 GABA(C) receptors. To investigate whether the inhibition of currents was due to a decrease in efficacy or in the potency of rho1 GABA(C) receptor, concentration-response curves for GABA were compared before and after PMA treatment. The EC50 concentrations of GABA obtained during the maximally inhibited period were not statistically different from the concentrations obtained before PMA treatment (1.74 +/- 0.33 and 1.45 +/- 0.28 microM, respectively). These results indicate that the inhibition depends on a change in number or conductance of active receptor channels, but not on a change in affinity for GABA. To allow histochemical detection of rho1 GABA(C) receptors, we constructed a receptor tagged at the C-terminal position with human c-myc epitope. Electrophysiologically, the tagged receptors showed almost the same sensitivities for GABA and PMA as those of wild-type rho1 GABA(C) receptors. Immunohistochemistry with anti-myc antibody detected a dense concentration of tagged receptors at the surface area of Xenopus oocytes. Transient exposure to PMA reduced the density of immunofluorescence at the surface area and increased it in the subsurface area. These results suggest that the stimulation of protein kinase C leads to internalization of rho1 GABA(C) receptors expressed in Xenopus oocytes.

Post-translational modification of alphaB-crystallin of normal human lens.

The current study reports several post-translational modifications of alphaB-crystallin in normal human lenses. The isoforms of post-translational modified alphaB-crystallin were isolated from the normal human lenses of >70-age group by ion exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoform modifications were determined in detail by fast atom bombardment mass spectroscopy and amino acid sequence analysis. As the criterion of non-modified alphaB-crystallin, alphaB-crystallin from 1-d-old infant lenses were used. The modifications found in this study involve oxidation of the N-terminal methionine-1 residue, phosphorylation of the serine-59 residue, and truncation of four amino acids from the C-terminal position of the crystallin. The oxidation of methionine-1 was found in the early stage of human life in 1-d-old lens, although other modification of alphaB-crystallin were usually only found in old lenses (>70-age group).

Utilization of the indirect lysosome targeting pathway by lysosome-associated membrane proteins (LAMPs) is influenced largely by the C-terminal residue of their GYXXphi targeting signals.

A systematic study was conducted on the requirements at the C-terminal position for the targeting of LAMPs to lysosomes, examining the hypothesis that a bulky hydrophobic residue is required. Mutations deleting or replacing the C-terminal valine with G, A, C, L, I, M, K, F, Y, or W were constructed in a reporter protein consisting of the lumenal/extracellular domain of avian LAMP-1 fused to the transmembrane and cytoplasmic domains of LAMP-2b. The steady-state distribution of each mutant form in mouse L-cells was assessed by quantitative antibody binding assays and immunofluorescence microscopy; efficiency of internalization from the plasma membrane and delivery to the lysosome were also estimated. It is found that (a) only C-terminal V, L, I, M, and F mediated efficient targeting to lysosomes, demonstrating the importance hydrophobicity and an optimal size of the C-terminal residue in targeting; (b) efficiency of lysosomal targeting generally correlated with efficiency of internalization; and (c) mutant forms that did not target well to lysosomes showed unique distributions in cells rather than simply default accumulation in the plasma membrane. Interactions of the targeting signals with adaptor subunits were measured using a yeast two-hybrid assay. The results are consistent with the hypothesis that trafficking of LAMP forms in cells through the indirect pathway is determined by the affinities of their targeting signals, predominantly for the mu2 and mu3 adaptors involved at plasma membrane and endosomal cellular sorting sites, respectively.

Decisive structural determinants for the interaction of proline derivatives with the intestinal H+/peptide symporter.

To elucidate the decisive structural factors relevant for dipeptide-carrier interaction, the affinity of short amide and imide derivatives for the intestinal H+/peptide symporter (PEPT1) was investigated by measuring their ability to inhibit Gly-Sar transport in Caco-2 cells. Dipeptides with proline or alanine in the C-terminal position displayed affinity constants (Ki) of 0.15-1.2 mM and 0.08-9.5 mM, respectively. There was no clear relationship between hydrophobicity, size or ionization status of the N-terminal amino acid and the affinity of the dipeptides. However, analyzing the individual peptide bond conformations of Xaa-Pro dipeptides, a striking correlation between the cis/trans ratios (trans contents 24-70%) and the affinity constants was observed. After correcting the Ki values for the incompetent cis isomers, the Ki corr values of most dipeptides were in a small range of 0.1-0.16 mM. This result revealed the decisive role of peptide bond conformation even for a transport protein that is quite promiscuous in substrate translocation. When measuring affinity constants of Xaa-Pro and Xaa-Sar dipeptides, the cis/trans ratios cannot be ignored. Lower affinities of Lys-Pro, Arg-Pro and Pro-Pro indicate that additional molecular factors affect their binding at PEPT1. The Ki values obtained for the corresponding Xaa-Ala dipeptides support this conclusion. Potential substrates or inhibitors of peptide transport were found among Xaa-piperidides and Xaa-thiazolidides. Dipeptides with N-terminal proline displayed a very diverse affinity profile. However, in contrast to current knowledge, several Pro-Xaa dipeptides such as Pro-Leu, Pro-Tyr and Pro-Pro are recognized by PEPT1 with appreciable affinities. Binding seems mainly determined by the hydrophobicity of the C-terminal amino acid and the rigidity of the structure.

Isolation and amino acid sequence of two trypsin isoinhibitors from duck pancreas

DPTI II and DPTI IV, two trypsin inhibitors from duck pancreas, have been isolated by affinity chromatography on immobilized anhydrotrypsin, anion exchange and RP-HPLC. The complete amino acid sequence of both inhibitors was determined after reductive carboxymethylation and digestion with Staphylococcus aureus V8 protease or trypsin. The inhibitors were each found to be a single polypeptide chain comprised of 69 amino acid residues and their molecular masses were estimated at 7687 Da for DPTI II and 7668 Da for DPTI IV. The only difference in amino acid sequence between the two inhibitors is the replacement of Arg for His residue in the C-terminal position of DPTI IV.

A side-reaction in the SPPS of Trp-containing peptides.

Syntheses of several Trp-containing peptides on a Wang solid support afforded significant amounts of a side-product. 1H-NMR and MS data showed that an unexpected alkylation by the linker has occurred on the indole nucleus. This was observed whatever the scavenger used, and whatever the position of the Trp residue in the sequence, unless it was in the C-terminal position.

Further Delineation of the Two Binding Sites (R*n) Associated with Tachykinin Neurokinin-1 Receptors Using [3-Prolinomethionine11]SP Analogues

Two binding sites are associated with neurokinin-1 substance P receptors in both transfected cells and mammalian tissues. To further delineate the interactions between the crucial C-terminal methionine of substance P and these two binding sites, we have incorporated newly designed constrained methionines, i.e. (2S, 3S)- and (2S,3R)-prolinomethionines. The potencies of these C terminus-modified SP analogues to bind both sites and to activate phosphatidylinositol hydrolysis and cAMP formation have been measured, together with those of their corresponding sulfoxides and sulfones. The molecular nature of these two binding sites and their selective coupling to effector signaling pathways are discussed in the light of current models of receptor activation. The less abundant binding site is coupled to G(q/11) proteins, whereas the most abundant one interacts with G(s) proteins in Chinese hamster ovary cells transfected with human neurokinin-1 receptors. The specific orientation of the C-terminal methionine side chain imposed by these constraints shows that macroscopically chi(1) and chi(2) angles of this crucial C-terminal residue are similar in both binding sites. However, slight but significant variations in the rotation around the Cgamma-S bond yield different either stabilizing or destabilizing interactions in the two binding sites. These results highlight the need of such constrained amino acids to probe subtle interactions in ligand-receptor complexes.

Diverse Fine Specificity and Receptor Repertoire of T Cells Reactive to the Major VP1 Epitope (VP1230–250) of Theiler’s Virus: Vβ Restriction Correlates with T Cell Recognition of the C-Terminal Residue

AbstractTheiler’s murine encephalomyelitis virus induces chronic demyelinating disease in genetically susceptible mice. The histopathological and immunological manifestation of the disease closely resembles human multiple sclerosis, and, thus, this system serves as a relevant infectious model for multiple sclerosis. The pathogenesis of demyelination appears to be mediated by the inflammatory Th1 response to viral epitopes. In this study, T cell repertoire reactive to the major pathogenic VP1 epitope region (VP1233–250) was analyzed. Diverse minimal T cell epitopes were found within this region, and yet close to 50% of the VP1-reactive T cell hybridomas used Vβ16. The majority (8/11) of the Vβ16+ T cells required the C-terminal amino acid residue on the epitope, valine at position 245, and every T cell hybridoma recognizing this C-terminal residue expressed Vβ16. However, the complementarity-determining region 3 sequences of the Vβ16+ T cell hybridomas were markedly heterogeneous. In contrast, such a restriction was not found in the Vα usage. Only restricted residues at this C-terminal position allowed for T cell activation, suggesting that Vβ16 may recognize this terminal residue. Further functional competition analysis for TCR and MHC class II-contacting residues indicate that many different residues can be involved in the class II and/or TCR binding depending on the T cell population, even if they recognize the identical minimal epitope region. Thus, recognition of the C-terminal residue of a minimal T cell epitope may associate with a particular Vβ (but not Vα) subfamily-specific sequence, resulting in a highly restricted Vβ repertoire of the epitope-specific T cells.

Human T cell recognition of the Mycobacterium leprae LSR antigen: epitopes and HLA restriction.

We have in this work mapped epitopes and HLA molecules used in human T cell recognition of the Mycobacterium leprae LSR protein antigen. HLA typed healthy subjects immunized with heat killed M. leprae were used as donors to establish antigen reactive CD4+ T cell lines which were screened for proliferative responses against overlapping synthetic peptides covering the C-terminal part of the antigen sequence. By using this approach we were able to identify two epitope regions represented by peptide 2 (aa 29-40) and peptide 6 (aa 49-60), of which the former was mapped in detail by defining the N- and C-terminal amino acid positions necessary for T cell recognition of the core epitope. MHC restriction analysis showed that peptide 2 was presented to T cells by allogeneic cells coexpressing HLA-DR4 and DRw53 or DR7 and DRw53. In contrast, peptide 6 was presented to T cells only in the context of HLA-DR5 molecules. In conclusion, the M. leprae LSR protein antigen can be recognized by human T cells in the context of multiple HLA-DR molecules, of which none are reported to be associated with the susceptibility to develop leprosy. The results obtained are in support of using the LSR antigen in subunit vaccine design.

Human T cell recognition of the Mycobacterium leprae LSR antigen: epitopes and HLA restriction

We have in this work mapped epitopes and HLA molecules used in human T cell recognition of the Mycobacterium leprae LSR protein antigen. HLA typed healthy subjects immunized with heat killed M. leprae were used as donors to establish antigen reactive CD4+ T cell lines which were screened for proliferative responses against overlapping synthetic peptides covering the C-terminal part of the antigen sequence. By using this approach we were able to identify two epitope regions represented by peptide 2 (aa 29–40) and peptide 6 (aa 49–60), of which the former was mapped in detail by defining the N- and C-terminal amino acid positions necessary for T cell recognition of the core epitope. MHC restriction analysis showed that peptide 2 was presented to T cells by allogeneic cells coexpressing HLA-DR4 and DRw53 or DR7 and DRw53. In contrast, peptide 6 was presented to T cells only in the context of HLA-DR5 molecules. In conclusion, the M. leprae LSR protein antigen can be recognized by human T cells in the context of multiple HLA-DR molecules, of which none are reported to be associated with the susceptibility to develop leprosy. The results obtained are in support of using the LSR antigen in subunit vaccine design.

A Continuous Fluorometric Assay for Leukotriene D4Hydrolase

A fluorogenic substrate for assay of leukotriene D4hydrolase (LTDase; EC 3.4.13.19) has been prepared and evaluated, using enzyme purified from porcine kidney. The compound is based on internal quenching of the synthetic, fluorescent amino acidd,l-2-amino-3-(7-methoxy-4-coumaryl)propanoic acid (d,l-Amp) by a 2,4-dinitrophenyl (DNP) group. The compound is ϵ-DNP-l-Lys-d-Amp which incorporates the D-isomer of Amp to exploit the unique ability among mammalian peptidases for LTDase to hydrolyze peptides containing ad-amino acid in the C-terminal position. ϵ-DNP-l-Lys-d-Amp was found to be an excellent substrate for LTDase, withKmvalue of 370 μM. Under the conditions of assay, the substrate was without noticeable quenching effect on the fluorescence of the product (d-Amp) liberated by the action of LTDase. Using porcine kidney microvillar membranes, which contain a battery of peptidases, the specific inhibitor of LTDase, cilastatin, completely inhibited the breakdown of ϵ-DNP-l-Lys-d-Amp, indicating that the substrate is selective for LTDase.

Strict Conservation of the Retroviral Nucleocapsid Protein Zinc Finger Is Strongly Influenced by Its Role in Viral Infection Processes: Characterization of HIV-1 Particles Containing Mutant Nucleocapsid Zinc-Coordinating Sequences

The retroviral nucleocapsid (NC) protein contains highly conserved amino acid sequences (-Cys-X2-Cys-X4-His-X4-Cys-) designated retroviral (CCHC) Zn2+fingers. The NC protein of murine leukemia viruses contains one NC Zn2+finger and mutants that were competent in metal binding (CCCC and CCHH) packaged wild-type levels of full-length viral RNA but were not infectious. These studies were extended to human immunodeficiency virus type 1 (HIV-1), a virus with two NC Zn2+fingers. Viruses with combinations of CCHC, CCCC, and CCHH Zn2+fingers in each position of HIV-1 NC were characterized. Mutant particles contained the normal complement of processed viral proteins. Four mutants packaged roughly wild-type levels of genomic RNA, whereas the remaining mutants packaged reduced levels. Virions with mutated C-terminal position NC fingers were replication competent. One interesting mutant, containing a CCCC Zn2+finger in the N-terminal position of NC, packaged wild-type levels of viral RNA and showed ∼5% wild-type levels of infectivity when examined in CD4-expressing HeLa cells containing an HIV-1 LTR/β-galactosidase construct. However, this particular mutant was replication defective in H9 cells; all other mutants were replication defective over the 8-week course of the assay. Two long terminal repeat viral DNA species could be detected in the CCCC mutant but not in any of the other replication-defective mutants. These studies show that the N-terminal Zn2+finger position is more sensitive to alterations than the C-terminal position with respect to replication. Additionally, the retroviral (CCHC) NC Zn2+finger is required for early infection processes. The evolutionary pressure to maintain CCHC NC Zn2+fingers depends mainly on its function in infection processes, in addition to its function in genome packaging.

DdlN from vancomycin-producing Amycolatopsis orientalis C329.2 is a VanA homologue with D-alanyl-D-lactate ligase activity.

Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in D-alanyl-D-lactate. A key enzyme in this process is a D-alanyl-D-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide D-alanyl-D-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism Amycolatopsis orientalis C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for D-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.