November 29
PROGRAM

12Glycoscience : diversity and integration

Cellular metabolism of natural andunnatural sialic acids
Jennifer Kohler, Ph.D.
Assistant Professor
Department of Biochemistry, L4.256B
University of Texas Southwestern Medical Center, USA

Jennifer Kohler
ProfileJennifer Kohler is a chemical biologist
studying glycobiology. She obtained herA.B. in Chemistry from Bryn Mawr College in 1994. She attended graduate schoolat Yale University, receiving her Ph.D. inChemistry in 2000. Her Ph.D. research wasconducted in the laboratory of Prof. Alanna Schepartz and focused on protein-DNAinteractions. From 2000-2004, Dr. Kohler
was an American Cancer Society postdoctoral fellow with Prof. Carolyn Bertozzi
at the University of California, Berkeley.
In the Bertozzi lab, she studied the function and modularity of Golgi-resident
glycosyltransferases. In 2005, Dr. Kohler
established an independent research groupin the Chemistry Department at Stanford
University and, in 2007, her group moved
to the University of Texas Southwestern
Medical Center in Dallas, Texas. Currently, she is an Assistant Professor in theDepartment of Biochemistry at Universityof Texas Southwestern Medical Center. Dr.
Kohler’s awards include the Alfred P. Sloan
Research Fellowship and the Camille andHenry Dreyfus New Faculty Award. Her
research group studies the metabolism ofmonosaccharides and the function of glycoconjugates, with particular emphaseson sialic acids and N-acetylglucosamine(GlcNAc). To conduct these studies, theresearch group employs a variety of experimental approaches, including chemical
synthesis, biochemistry, and cell biology.

sialic acid, diazirine,
Keywords photocrosslinking, gangliosides,
toxins42

Sialic acid is a critical component of many
mammalian glycans and serves a variety of roles. Sialylated glycoconjugatesmediate essential recognition events in
the immune system and also act as receptors for many pathogens and their toxins.
Sialylated molecules are abundant in the
nervous system, where they coordinate
cell-cell interactions. Finally, many recognized tumor markers are composed of
sialylated molecules.
Given the critical roles of sialic acid, it
is intriguing to note that humans differ
from other primates in certain aspects ofsialic acid biology. Notably, humans areincapable of synthesizing a hydroxylated
version of sialic acid known as N-glycolylneuraminic acid (NeuGc) and make only
the non-hydroxylated form, N-acetylneuraminic acid (NeuAc). The NeuGc
deficiency in humans is attributed to atruncation in the gene encoding cytidine
monophosphate sialic acid hydroxylase(CMAH). Despite their inability to make
NeuGc, humans can incorporate this sialic
acid into glycoconjugates through dietaryacquisition. These NeuGc-containing
glycoconjugates can be immunogenic inhumans and are implicated in chronic
inflammatory states, including those associated with cancer progression. Moreover,the presence of NeuGc-containing glycoconjugates predisposes humans to infectious diseases where the pathogens or toxinbind to NeuGc. Because the incorporationof NeuGc carries negative consequences
for humans, we hypothesized that humancells may employ mechanisms to discriminate against NeuGc incorporation.
To track the metabolism of NeuAc andNeuGc, we prepared cell-permeable formsof N-acetylmannosamine (ManNAc) andN-glycolylmannosamine (ManNGc).
ManNAc and ManNGc serve as metabolic
precursors to NeuAc and NeuGc. We discovered that both human and rodent celllines preferentially metabolize ManNAc toNeuAc-containing glycoconjugates, whileless efficiently metabolizing ManNGc

the 20th Anniversary of the Mizutani Foundation for Glycoscienceto NeuGc-containing glycoconjugates.
Discrimination appears to occur during
several of the required enzymatic transformations. Notably, the rodent cell line wasmore efficient at metabolizing ManNGc
than the human cell line was. Our results
suggest that both humans and rodents
may be under evolutionary pressure toexclude NeuGc from sialylated molecules,
but that this pressure may be stronger inhumans. Our current work in this area
focuses on examining the NeuAc/NeuGc
specificity of individual sialyltransferases,
the enzymes that add sialic acids to glycoconjugates. Future work will delineate thefunctions of those sialylated products andreveal why they might be subject to evolutionary forces.
In addition to examining the metabolism of naturally occurring sialylated
molecules, we also exploit the sialic acidmetabolic pathway to produce unnatural
sialylated molecules. In particular, wedeveloped a method to produce sialylated
molecules that contain the diazirine photocrosslinking group. In this method,mammalian cell lines are cultured with acell-permeable form of ManNAc, which
has been chemically modified to include
an N-acyl diazirine substituent. Cells
metabolize the diazirine-modified ManNAc compound to diazirine-modified
sialic acid (SiaDAz) and then add thissialic acid analog to glycoproteins and glycolipids that are displayed on the cell surface. The diazirine can be activated with
UV radiation, producing a highly reactive
carbene. The carbene then inserts intoneighboring molecules, forming a covalent bond between the sialylated moleculeand its binding partner. These crosslinked
complexes can be isolated and characterized using immunoblots or mass spectrometry analysis. In a proof-of-principle
experiment, we demonstrated that a sialic
acid binding Ig-like lectin, CD22, could be
photocrosslinked to sialylated molecules,
which include additional copies of CD22.
Currently, we are using metabolicallyFigure 1. Metabolic incorporation of natural and unnatural sialic acids.
A NeuAc and NeuGc are two common forms of sialic acid. Cells cultured with cell-permeable metabolic precursors of NeuAcand NeuGc preferentially display NeuAc on the cell surface.
B Metabolic engineering enables the introduction of the diazirine photocrosslinker onto sialylated molecules displayed on
the surface of mammalian cells. UV radiation activates the diazirine, resulting in covalent crosslinking between sialylated
molecules and their binding partners.

incorporated SiaDAz to discover the interaction partners of sialylated proteins andthe substrates of sialidases, enzymes thatremove sialic acid.
We also demonstrated that SiaDAz is
incorporated into gangliosides, sialic
acid-containing glycoconjugates that arefound in all animal tissues. We investigated whether metabolically incorporated SiaDAz could be used to covalently

crosslink a ganglioside-protein complex.
The ganglioside GM1a is recognized to be
the receptor used by cholera toxin to bindto and invade host intestinal epithelialcells. We induced Jurkat cells to produce
SiaDAz-containing gangliosides by culturing these cells with the cell-permeable
form of diazirine-modified ManNAc. The
cells were incubated with the B subunitof cholera toxin (CTxB) and irradiatedto induce crosslinking. We observed effi-cient formation of a crosslinked complex
containing GM1a and CTxB, which we
characterized by immunoblot and mass
spectrometry. Current efforts focus on
discovering additional binding partners
for cholera toxin and other toxins, using
metabolic incorporation of SiaDAz in celllines that mimic the physiological sites ofintoxication.

References1) 	Tanaka Y, Kohler, JJ. Photoactivatable crosslinking sugars for capturing glycoprotein interactions, J. Amer. Chem. Soc. (130):3278-3279, 2008.
2) 	Bond MR, Zhang H, Vu PD, Kohler JJ. Photocrosslinking of glycoconjugatesusing metabolically incorporated diazirine-containing sugars, Nature Prot.
(4):1044-1063, 2009.
3) 	Bond MR, Whitman CM, Kohler JJ. Metabolically incorporated photocrosslinking sialic acid covalently captures a ganglioside-protein complex, Mol.

Biosys. (6):1796-1799, 2010.
4) 	Whitman CM, Yang F, Kohler JJ. Modified GM3 gangliosides produced bymetabolic oligosaccharide engineering, Bioorg. Med. Chem. Lett. (21): 50065010, 2011.
5) 	Bond MR, Zhang H, Kim J, Yu S-H, Yang F, Patrie SM, Kohler JJ. Metabolismof diazirine-modified N-acetylmannosamine analogs to photocrosslinking
sialosides, Bioconj. Chem. (22): 1811-1823, 2011.

Commemorative Symposium43