Characterization of ligand binding to the bifunctional key enzyme in the sialic acid biosynthesis by NMR: II. Investigation of the ManNAc kinase functionality

N-Acetylmannosamine (ManNAc) is the physiological precursors to all sialic acids that occur in nature. As variations in the sialic acid decoration of cell surfaces can profoundly affect cell-cell, pathogen-cell, or drug-cell interactions, the enzymes that convert ManNAc into sialic acid are attractive targets for the development of drugs that specifically interrupt sialic acid biosynthesis or lead to modified sialic acids on the surface of cells. The first step in the enzymatic conversion of ManNAc into sialic acid is phosphorylation, yielding N-acetylmannosamine-6-phosphate. The enzyme that catalyzes this conversion is the N-acetylmannosamine kinase (ManNAc kinase) as part of the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Here, we employed saturation transfer difference (STD) NMR experiments to study the binding of ManNAc and related ligands to the ManNAc kinase. It is shown that the configuration of C1 and C4 of ManNAc is crucial for binding to the enzyme, whereas the C2 position not only accepts variations in the attached N-acyl side chain but also tolerates inversion of configuration. Our experiments also show that ManNAc kinase maintains its functionality, even in the absence of Mg(2+). From the analysis of the STD NMR-derived binding epitopes, it is concluded that the binding mode of the N-acylmannosamines critically depends on the N-acyl side chain. In conjunction with the relative binding affinities of the ligands obtained from STD NMR titrations, it is possible to derive a structure-binding affinity relationship. This provides a cornerstone for the rational design of drugs for novel therapeutic applications by altering the sialic acid decorations of cell walls.     

Stimulation of human peripheral blood mononuclear cells by the sialic acid precursor N-propanoylmannosamine

N-Propanoylmannosamine is an unnatural precursor of sialic acid, which is taken up by a variety of animal cells and metabolized to N-propanoylneuraminic acid. In several studies it has been demonstrated that application of unnatural precursors of sialic acids such as N-propanoylmannosamine (ManNProp) and homologues interfere with cell differentiation and proliferation of neuronal cells or embryonic stem cells. Since the function of the immune system is known to rely on the presence of sialic acid, we applied ManNProp to human peripheral blood mononuclear cells (PBMC). When culturing those lymphocytes with ManNProp 10 % of the natural sialic acid N-acetylneuraminic acid could be replaced by the newly formed N-propanoylneuraminic acid. This procedure resulted (a) in a marked stimulation in the rate of proliferation of PBMC, (b) a 10-fold increase of IL-2 production coupled with an up-regulation of its receptor CD25 on the cell surface and (c) a concomitant expression and regulation of the transferrin receptor with cell growth. The stimulation of PBMC by ManNProp might therefore introduce a new approach of immunomodulation.     

Glycoengineering the N-acyl side chain of sialic acid of human erythropoietin affects its resistance to sialidase

Abstract During the last years, the use of therapeutic glycoproteins has increased strikingly. Glycosylation of recombinant glycoproteins is of major importance in biotechnology, as the glycan composition of recombinant glycoproteins impacts their pharmacological properties. The terminal position of N-linked complex glycans in mammals is typically occupied by sialic acid. The presence of sialic acid is crucial for functionality and affects the half-life of glycoproteins. However, glycoproteins in the bloodstream become desialylated over time and are recognized by the asialoglycoprotein receptors via the exposed galactose and targeted for degradation. Non-natural sialic acid precursors can be used to engineer the glycosylation side chains by biochemically introducing new non-natural terminal sialic acids. Previously, we demonstrated that the physiological precursor of sialic acid (i.e., N-acetylmannosamine) can be substituted by the non-natural precursors N-propanoylmannosamine (ManNProp) or N-pentanoylmannosamine (ManNPent) by their simple application to the cell culture medium. Here, we analyzed the glycosylation of erythropoietin (EPO). By feeding cells with ManNProp or ManNPent, we were able to incorporate N-propanoyl or N-pentanoyl sialic acid in significant amounts into EPO. Using a degradation assay with sialidase, we observed a higher resistance of EPO to sialidase after incorporation of N-propanoyl or N-pentanoyl sialic acid.     

Biosynthesis of a nonphysiological sialic acid in different rat organs, using N-propanoyl-D-hexosamines as precursors.

In this study it could be shown that in rat the normally occurring N-acetyl neuraminic acid can be modified in its N-acyl moiety by in vivo administration of the chemically synthesized N-propanoyl precursors, N-propanoyl-D-glucosamine or N-propanoyl-D-mannosamine. It could be shown that each of these nonphysiological amino sugar analogues was incorporated into both membrane and serum glycoproteins. After treatment of rats with radiolabeled N-[acyl-1-14C]D-mannosamine, radioactivity could be removed from serum glycoprotein fractions by incubation with neuraminidase from Clostridium perfringens or from Arthrobacter ureafaciens. Mild acid hydrolysis removed 98% of the radioactivity after in vivo labeling with N-[acetyl-1-14C]D-mannosamine and 86% after labeling with N-[propanoyl-1-14C]D-mannosamine. Chromatographic analysis yielded two compounds, i.e. N-acetyl neuraminic acid and N-propanoyl neuraminic acid, the latter being identified by gas liquid chromatography/mass spectrometry studies. Measurement of protein-bound radioactivity in different rat organs revealed a different organotropy of the natural and the nonphysiological neuraminic acid precursor. Of the glucosamine derivatives, N-acetyl-D-glucosamine showed the higher rate of uptake and incorporation in most organs (except in the submandibulary gland), and especially in kidney cortex and Morris hepatoma 7777. Natural and the unphysiological mannosamine derivatives were incorporated at similar rates, except in liver, where N-acetyl-D-mannosamine was taken up and metabolized more effectively. This finding indicates that it is possible to modify the acyl group of N-acetyl neuraminic acid in vivo by the introduction of an N-propanoyl group and possibly other homologous N-acyl groups. This procedure may provide a tool for a further characterization of the biological function of sialic acids.     

Substrate specificity of the sialic acid biosynthetic pathway.

Unnatural analogues of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogues bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogues with ketone-containing N-acyl groups that varied in the length or steric bulk was chemically synthesized and tested for metabolic conversion to cell surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.     

Consequences of a subtle sialic acid modification on the murine polyomavirus receptor.

Polyomaviruses are small, nonenveloped DNA tumor viruses with restricted host ranges. Virus binding to cell surface receptors is one determinant of viral tropism. Although murine polyomavirus is among the best characterized viruses, little is known about the sialic acid-containing receptor and its interaction with viral particles. By using nonradioactive virus binding assays as recently described for the B-lymphotropic papovavirus, murine polyomavirus particles were found to bind in a saturable and noncooperative manner to 25,000 receptors per 3T6 mouse fibroblast. The virus-receptor interaction at 4 degrees C was of high affinity (Kd = 1.8 x 10(-11) M), very fast (k1 = 1.7 x 10(7) M(-1) s(-1)), and stable (half-life = 38 min). Elongation of the N-acyl side chain of sialic acid by biosynthetic modulation with synthetic precursor analogs has been shown for other polyomaviruses to influence both sialic acid-dependent binding and infection (O. T. Keppler, P. Stehling, M. Herrmann, H. Kayser, D. Grunow, W. Reutter, and M. Pawlita, J. Biol. Chem. 270:1308-1314, 1995). In 3T6 cells in which about one-third of the sialic acids were modified, infection and binding of polyomavirus particles were significantly reduced. The number of receptors per cell was decreased to 18,000, with the remaining receptors displaying the same affinity as in untreated cells. Molecular modeling studies based on the three-dimensional structure of a mouse polyomavirus-sialyllactose complex recently solved by T. Stehle and coworkers (T. Stehle, Y. W. Yan, T. L. Benjamin, and S. C. Harrison, Nature 369:160-163, 1994) were performed. They suggest that the elongation of the N-acyl side chain by a single methylene group leads to steric hinderence, with the peptide backbone of a loop walling the tip of the shallow sialic acid binding groove. This collision appears to be incompatible with functional binding. The data are taken as a basis to discuss possible features of the organization and topology of the cellular receptor for mouse polyomavirus.     

Engineering sialic acid synthetic ability into insect cells: identifying metabolic bottlenecks and devising strategies to overcome them

Previous studies have indicated negligible levels of both sialylation and the precursor N-acetylneuraminic acid (Neu5Ac) in a number of insect cell lines grown in serum-free medium. The overexpression of the human sialic acid 9-phosphate synthase (SAS) in combination with N-acetylmannosamine (ManNAc) feeding has been shown to overcome this limitation. In this study we evaluated the potential bottlenecks in the sialic acid synthesis pathway in a Spodoptera frugiperda (Sf9) insect cell line and devised strategies to overcome them by overexpression of the enzymatic pathway enzymes combined with appropriate substrate feeding. Coexpression of SAS and UDP-GlcNAc 2-epimerase/ManNAc kinase, the bifunctional enzyme initiating sialic acid biosynthesis in mammals, resulted in Neu5Ac synthesis without use of any external media supplementation to demonstrate that Neu5Ac could be generated intracellularly in Sf9 cells using natural metabolic precursors. N-Acetylglucosamine (GlcNAc) feeding in combination with this coexpression resulted in much higher levels of Neu5Ac compared to levels obtained with ManNAc feeding with SAS expression alone. The lower Neu5Ac levels obtained with ManNAc feeding suggested limitations in the transport and phosphorylation of ManNAc. The bottleneck in phosphorylation was likely due to utilization of GlcNAc kinase for phosphorylation of ManNAc in insect cells and was overcome by expression of ManNAc kinase. The transport limitation was addressed by the addition of tetra-O-acetylated ManNAc, which is easily taken up by the cells. An alternative sialic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), could also be generated in insect cells, suggesting the potential for controlling not only the production of sialic acids but also the type of sialic acid generated. The levels of KDN could be increased with virtually no Neu5Ac generation when Sf9 cells were fed excess GlcNAc. The results of these studies may be used to enhance the sialylation of target glycoproteins in insect and other eukaryotic expression systems.     

Characterization of the cellular uptake and metabolic conversion of acetylated N-acetylmannosamine (ManNAc) analogues to sialic acids

"Sialic acid engineering" refers to the strategy where cell surface carbohydrates are modified by the biosynthetic incorporation of metabolic intermediates, such as non-natural N-acetylmannosamine (ManNAc) analogues, into cellular glycoconjugates. While this technology has promising research, biomedical, and biotechnological applications due to its ability to endow the cell surface with novel physical and chemical properties, its adoption on a large scale is hindered by the inefficient metabolic utilization of ManNAc analogues. We address this limitation by proposing the use of acetylated ManNAc analogues for sialic acid engineering applications. In this paper, the metabolic flux of these "second-generation" compounds into a cell, and, subsequently, into the target sialic acid biosynthetic pathway is characterized in detail. We show that acetylated ManNAc analogues are metabolized up to 900-fold more efficiently than their natural counterparts. The acetylated compounds, however, decrease cell viability under certain culture conditions. To determine if these toxic side effects can be avoided, we developed an assay to measure the cellular uptake of acetylated ManNAc from the culture medium and its subsequent flux into sialic acid biosynthetic pathway. This assay shows that the majority ( > 80%) of acetylated ManNAc is stored in a cellular "reservoir" capable of safely sequestering this analogue. These results provide conditions that, from a practical perspective, enable the acetylated analogues to be used safely and efficaciously and therefore offer a general strategy to facilitate metabolic substrate-based carbohydrate engineering efforts. In addition, these results provide fundamental new insights into the metabolic processing of non-natural monosaccharides.     

Biosynthetic incorporation of unnatural sialic acids into polysialic acid on neural cells

In this study we demonstrate that polysialyltransferases are capable of accepting unnatural substrates in terminally differentiated human neurons. Polysialyltransferases catalyze the glycosylation of the neural cell adhesion molecule (NCAM) with polysialic acid (PSA). The unnatural sialic acid analog, N-levulinoyl sialic acid (SiaLev), was incorporated into cell surface glycoconjugates including PSA by the incubation of cultured neurons with the metabolic precursor N-levulinoylmannosamine (ManLev). The ketone group within the levulinoyl side chain of SiaLev was then used as a chemical handle for detection using a biotin probe. The incorporation of SiaLev residues into PSA was demonstrated by protection from sialidases that can cleave natural sialic acids but not those bearing unnatural N-acyl groups. The presence of SiaLev groups on the neuronal cell surface did not impede neurite outgrowth or significantly affect the distribution of PSA on neuronal compartments. Since PSA is important in neural plasticity and development, this mechanism for modulating PSA structure might be useful for functional studies.     

Improvement of interferon-gamma sialylation in Chinese hamster ovary cell culture by feeding of N-acetylmannosamine

Because the presence of sialic acid can extend circulatory lifetime, a high degree of sialylation is often a desirable feature of therapeutic glycoproteins. In this study, the incomplete intracellular sialylation of interferon-gamma (IFN-gamma), produced by Chinese hamster ovary cell culture, was minimized by supplementing the culture medium with N-acetylmannosamine (ManNAc), a direct intracellular precursor for sialic acid synthesis. By introducing 20 mM ManNAc into the culture medium, incompletely sialylated biantennary glycan structures were reduced from 35% to 20% at the Asn97 glycosylation site. This effect was achieved without affecting cell growth or product yield. The intracellular pool of CMP-sialic acid, the nucleotide sugar substrate for sialyltransferase, was also extracted and quantified by HPLC. Feeding of 20 mM ManNAc increased this intracellular pool of CMP-sialic acid by nearly thirtyfold compared with unsupplemented medium. When radiolabeled ManNAc was used to trace the incorporation of the precursor, it was found that supplemental ManNAc was exclusively incorporated into IFN-gamma as sialic acid and that, at 20 mM ManNAc feeding, nearly 100% of product sialylation originated from the supplemental precursor.     

Increased expression of the selectin ligand sialyl-Lewis(x) by biochemical engineering of sialic acids

Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis of several diseases. The precursor of all physiological sialic acids is N-acetyl-d-mannosamine. Using N-propanoyl mannosamine, a novel precursor of sialic acid, we showed earlier that sialic acids with a prolonged N-acyl side chain (e.g., N-propanoyl neuraminic acid) are incorporated into cell surface glycoconjugates. In this study, we report the structural and functional consequences of the incorporation of the nonphysiological sialic acid, N-propanoyl neuraminic acid, into glycoconjugates of HL60-I cells. These cells do not express UDP-GlcAc-2-epimerase, the key enzyme of the biosynthesis of N-acetyl-d-mannosamine. Therefore, they do not express sialyl-Lewis(x) structures and consequently do not bind to selectins. Application of N-acetyl-d-mannosamine leads to the expression of sialyl-Lewis(x) structures and to binding to selectins. Surprisingly, incorporation of N-propanoyl neuraminic acid into glycoconjugates of these cells leads to a dramatic increase of sialyl-Lewis(x) structures and to increased adhesion to selectins.     

Efficient biochemical engineering of cellular sialic acids using an unphysiological sialic acid precursor in cells lacking UDP-N-acetylglucosamine 2-epimerase

Sialic acids comprise a family of terminal sugars essential for a variety of biological recognition systems. N-Propanoylmannosamine, an unphysiological sialic acid precursor, is taken up and metabolized by mammalian cells resulting in oligosaccharide-bound N-propanoylneuraminic acid. N-Propanoylmannosamine, applied to endogenously hyposialylated subclones of the myeloid leukemia HL60 and of the B-cell lymphoma BJA-B, both deficient in UDP-N-acetylglucosamine 2-epimerase, is efficiently metabolized to CMP-N-propanoylneuraminic acid resulting in up to 85% of glycoconjugate-associated sialic acids being unphysiological N-propanoylneuraminic acid. Thus, UDP-N-acetylglucosamine 2-epimerase-deficient cell lines provide an important experimental progress in engineering cells to display an almost homogeneous population of defined, structurally altered sialic acids.     

Synthesis of non-natural ManNAc analogs for the expression of thiols on cell-surface sialic acids

The sialic acid biosynthetic pathway in mammalian cells utilizes N-acetyl-D-mannosamine (ManNAc) as a natural metabolic precursor and has the remarkable ability to biosynthetically process non-natural ManNAc analogs. Herein, we describe a recipe-style protocol for the synthesis of the novel peracetylated analog Ac5ManNTGc (1) that contains a pendant acetylthio- group and enables incorporation of thiol functionalities into the glycocalyx of living cells. We also describe the synthesis of the oxygen analog Ac5ManNGc (2), which serves as an appropriate control compound for biological experiments with 1. Both 1 and 2 were prepared from a reported, common intermediate 8, which is selectively acetylated at the hydroxyl groups. In contrast to previous methods, this synthetic approach introduces O-acetyl groups first, followed by N-acylation. Starting from the commercially available D-mannosamine hydrochloride (5), gram quantities of both 1 and 2 can be prepared over five steps in about 2-3 weeks.     

Characterization of the metabolic flux and apoptotic effects of O-hydroxyl- and N-acyl-modified N-acetylmannosamine analogs in Jurkat cells

The supplementation of the sialic acid biosynthetic pathway with exogenously supplied N-acetylmannosamine (ManNAc) analogs has many potential biomedical and biotechnological applications. In this work, we explore the structure-activity relationship of Man-NAc analogs on cell viability and metabolic flux into the sialic acid biosynthetic pathway to gain a better understanding of the fundamental biology underlying "glycosylation engineering" technology. A panel of ManNAc analogs bearing various modifications on the hydroxyl groups as well as substitutions at the N-acyl position was investigated. Increasing the carbon chain length of ester derivatives attached to the hydroxyl groups increased the metabolic efficiency of sialic acid production, whereas similar modification to the N-acyl group decreased efficiency. In both cases, increases in chain length decreased cell viability; DNA ladder formation, Annexin V-FITC two-dimensional flow cytometry assays, caspase-3 activation, and down-regulation of sialoglycoconjugate-processing enzymes established that the observed growth inhibition and toxicity resulted from apoptosis. Two of the panel of 12 analogs tested, specifically Ac(4)ManNLev and Ac(4) ManNHomoLev, were highly toxic. Interestingly, both of these analogs maintained a ketone functionality in the same position relative to the core monosaccharide structure, and both also inhibited flux through the sialic acid pathway (the remainder of the less toxic analogs either increased or had no measurable impact on flux). These results provide fundamental insights into the role of sialic acid metabolism in apoptosis by demonstrating that ManNAc analogs can modulate apoptosis both indirectly via hydroxylgroup effects and directly through N-acyl-group effects.     

Accessibility of N-acyl-D-mannosamines to N-acetyl-D-neuraminic acid aldolase

N-Acetyl-D-neuraminic acid (NeuNAc) aldolase is an important enzyme for the metabolic engineering of cell-surface NeuNAc using chemically modified D-mannosamines. To explore the optimal substrates for this application, eight N-acyl derivatives of D-mannosamine were prepared, and their accessibility to NeuNAc aldolase was quantitatively investigated. The N-propionyl-, N-butanoyl-, N-iso-butanoyl-, N-pivaloyl-, and N-phenylacetyl-D-mannosamines proved to be as good substrates as, or even better than, the natural N-acetyl-D-mannosamine, while the N-trifluoropropionyl and benzoyl derivatives were poor. It was proposed that the electronic effects might have a significant influence on the enzymatic aldol condensation reaction of D-mannosamine derivatives, with electron-deficient acyl groups having a negative impact. The results suggest that N-propionyl-, N-butanoyl-, N-iso-butanoyl-, and N-phenylacetyl-D-mannosamines may be employed to bioengineer NeuNAc on cells.     

Metabolism of diazirine-modified N-acetylmannosamine analogues to photo-cross-linking sialosides

Terminal sialic acid residues often mediate the interactions of cell surface glycoconjugates. Sialic acid-dependent interactions typically exhibit rapid dissociation rates, precluding the use of traditional biological techniques for complex isolation. To stabilize these transient interactions, we employ a targeted photo-cross-linking approach in which a diazirine photo-cross-linker is incorporated into cell surface sialylated glycoconjugates through the use of metabolic oligosaccharide engineering. We describe three diazirine-modified N-acetylmannosamine (ManNAc) analogues in which the length of the linker between the pyranose ring and the diazirine was varied. These analogues were each metabolized to their respective sialic acid counterparts, which were added to both glycoproteins and glycolipids. Diazirine-modified sialic acid analogues could be incorporated into both α2-3 and α2-6 linkages. Upon exposure to UV irradiation, diazirine-modified glycoconjugates were covalently cross-linked to their interaction partners. We demonstrate that all three diazirine-modified analogues were capable of competing with endogeneous sialic acid, albeit to varying degrees. We found that larger analogues were less efficiently metabolized, yet could still function as effective cross-linkers. Notably, the addition of the diazirine substituent interferes with metabolism of ManNAc analogues to glycans other than sialosides, providing fidelity to selectively incorporate the cross-linker into sialylated molecules. These compounds are nontoxic and display only minimal growth inhibition at the concentrations required for cross-linking studies. This report provides essential information for the deployment of photo-cross-linking analogues to capture and study ephemeral, yet essential, sialic acid-mediated interactions.     

Polysialic acid bioengineering of neuronal cells by N-acyl sialic acid precursor treatment

The inherent promiscuity of the polysialic acid (PSA) biosynthetic pathway has been exploited by the use of exogenous unnatural sialic acid precursor molecules to introduce unnatural modifications into cellular PSA, and has found applications in nervous system development and tumor vaccine studies. The sialic acid precursor molecules N-propionyl- and N-butanoyl-mannosamine (ManPr, ManBu) have been variably reported to affect PSA biosynthesis ranging from complete inhibition to de novo production of modified PSA, thus illustrating the need for further investigation into their effects. In this study, we have used a monoclonal antibody (mAb) 13D9, specific to both N-propionyl-PSA and N-butanoyl-PSA (NPrPSA and NBuPSA), together with flow cytometry, to study precursor-treated tumor cells and NT2 neurons at different stages of their maturation. We report that both ManPr and ManBu sialic acid precursors are metabolized and the resultant unnatural sialic acids are incorporated into de novo surface sialylglycoconjugates in murine and human tumor cells and, for the first time, in human NT2 neurons. Furthermore, neither precursor treatment deleteriously affected endogenous PSA expression; however, with NT2 cells, PSA levels were naturally downregulated as a function of their maturation into polarized neurons independent of sialic acid precursor treatment.     

Modified GM3 gangliosides produced by metabolic oligosaccharide engineering

Metabolic oligosaccharide engineering is powerful approach to altering the structure of cellular sialosides. This method relies on culturing cells with N-acetylmannosamine (ManNAc) analogs that are metabolized to their sialic acid counterparts and added to glycoproteins and glycolipids. Here we employed two cell lines that are deficient in ManNAc biosynthesis and examined their relative abilities to metabolize a panel of ManNAc analogs to sialosides. In addition to measuring global sialoside production, we also examined biosynthesis of the sialic acid-containing glycolipid, GM3. We discovered that the two cell lines differ in their ability to discriminate among the variant forms of ManNAc. Further, our data suggest that modified forms of sialic acid may be preferentially incorporated into certain sialosides and excluded from others. Taken together, our results demonstrate that global analysis of sialoside production can obscure sialoside-specific differences. These findings have implications for downstream applications of metabolic oligosaccharide engineering, including imaging and proteomics.     

Modulation of cIAP-1 by novel antitubulin agents when combined with bryostatin 1 results in increased apoptosis in the human early pre-B acute lymphoblastic leukemia cell line Reh

Derivatives of N-acyl-D-mannosamine differing in the N-acyl-side chain can be metabolically converted into neuraminic acids with corresponding N-acyl side chains. In the present study we show the in vivo modulation of sialic acids in membrane-bound dipeptidyl peptidase IV (CD 26) from rat liver after administration of N-propanoyl-D-mannosamine. Treatment of rats with this unphysiological precursor resulted in an incorporation of N-propanoylneuraminic acid into N-linked glycans of dipeptidyl peptidase IV.