Creation of New Chondroitin Lyases Through Novel Methods Andrew Harper*, Mark J. Olsen Department of Mathematics, Chemistry, and Physics West Texas A

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Creation of New Chondroitin Lyases Through Novel
Methods Andrew Harper, Mark J. OlsenDepartment
of Mathematics, Chemistry, and PhysicsWest Texas
A M University Canyon, TX 79015

• Abstract
• Chondroitin sulfates are sugars known to carry
  information through their sulfation patterns.
  This plays important roles in many processes,
  such as cancer and stem cell differentiation. A
  major hurdle to deciphering and using this
  information is the lack of specific enzymes
  available to cut these sugars. This project will
  attempt to make a generalist enzyme
  (chondroitinase ABC I) that cuts many types of
  chondroitin subunits into a specialist enzyme
  that will cut only the specific, rare, and
  important chondroitin D subunit. The substrate
  selectivity of the entire enzyme will be changed
  only by altering the substrate binding domain. By
  using this method, this project will answer
  questions about the effect of substrate binding
  on selectivity as well as providing new tools for
  studying such sugars. This is a novel application
  of the established technique of directed
  evolution. A library of substrate binding domain
  mutants will be made by introducing random point
  mutations at a controlled rate. Changes in enzyme
  activity will be measured by display on yeast
  cell surfaces and measuring changes in cell
  fluorescence as the enzyme cuts chondroitin
  sulfates attached to fluorescent compounds which
  then adhere to the cell surface.

N-acetyl-galactosamine Uronate
Chondroitin A N-Acetyl-Galactosamine-4-sulfate Glucuronate
Chondroitin B N-Acetyl-Galactosamine-4,6-disulfate Glucuronate-2-sulfateorIduronate-2-sulfate
Chondroitin C N-Acetyl-Galactosamine-6-sulfate Glucuronate
Chondroitin D N-Acetyl-Galactosamine-6-sulfate Glucuronate-2-sulfate
Chondroitin E N-Acetyl-Galactosamine-4,6-disulfate Glucuronate
Introduction Chondroitin sulfates are natural
polysaccharides found in the extracellular
matrix. They are not only altered during tumor
progression and metastasis but also directly
affect wound healing, immunological responses,
stem cell development, stem cell maturation,
neurological development, and neuron growth.
Chondroitin sulfates are divided into
disaccharide subunit classifications based on
differences in sulfation and epimerization. Each
disaccharide subunit consists of an
N-acetyl-galactosamine residue and a uronate
residue and is given a letter designation, as
seen in the table. The use of different subunits
is thought to carry information and several
studies have shown that subunit differences
affect protein recognition of these
polysaccharides. Chondroitin ABC lyase I
(chondroitinase ABC or cABC) from Proteus
vulgaris is an enzyme capable of cleaving many
different kinds of these subunits, apparently
through the use of a necessary substrate binding
domain. It is a very useful and promising enzyme,
but has various flaws depending on the intended
application. For example, cABC has already been
very successful in improving the rate and degree
of recovery from spinal and central nervous
system injury in mice, even though it has poor
thermostability and is known to have low activity
against chondroitin D subunits, which are
important in various neurological processes.
Chondroitin ABC lyase I is one of several
chondroitin lyases useful in chondroitin sulfate
analysis, and while it is useful in some
situations due to its broad substrate
selectivity, there is a great need for more
selective chondroitin lyases for analytical
purposes. For example, there is no known
chondroitin lyase that selectively cuts
previously mentioned chondroitin D subunits.
Given the known strengths of cABC, it appears to
be a likely predecessor for the creation of a
more powerful generation of analytical and
therapeutic enzymes. A simple, elegant, and
direct approach to the engineering of this
protein is to control the selectivity of the
substrate binding/presentation domain through
directed evolution.
Methods The nucleotide sequence coding for the
n-terminal domain (i.e. the substrate binding
domain) of cABC has been displayed on the surface
of the yeast using the vector pCTCON. Mutant
libraries of the protein will be created using
error-prone PCR. Yeast cells will be
fluorescently labeled to determine the substrate
binding affinity and level of protein expression.
Fluorescently labeled peptides will be conjugated
to chondroitin sulfates of known subunits such
that the labeled polypeptides will be bound with
the substrate to the n-terminal domain. This
means that the more tightly a protein binds a
specific chondroitin type, the more fluorophores
will be bound to the cell expressing that
protein, causing the cell to appear more
fluorescent at that wavelength. Protein
expression will be determined using a
fluorescent, antibody-based system integrated
into the vector. Cells with the desired
fluorescence (and hence the desired protein
expression and substrate binding affinity) will
be retrieved from the library using a
fluorescence activated cell sorter (FACS). The
nucleotide sequence of the altered n-terminal
domain will be retrieved from these cells and
sequenced. After determining the effect of the
changes once the domain has been reintegrated
into the whole enzyme, the sequence may be used
as the template for the next generation of
libraries or may be considered a successful end
point.
Above left Chondroitin ABC lyase I from Proteus
vulgarisAbove right The substrate binding
domain from chondroitin ABC lyase I Below The
cycle of protein evolution through directed
evolution using random mutagenesis and
fluorescent activated cell sorting
Results, Conclusions, Future Directions The
chondroitin lyase ABC I gene has been extracted
from P. vulgaris and the n-terminal domain has
been integrated into the yeast cell surface
display vector. The next steps are to verify
protein expression and substrate binding. After
that, we will begin generation of libraries. The
entire chondroitinase ABC gene has been already
been inserted into the pCTCON vector, and in the
future will be engineered for thermostability.
Acknowledgements Research Corporation Killgore
Research Enhancement Grant USDA Seed Grant Dr.
James Hallmark

• Objectives
• The creation of a selective chondroitinase D from
  chondroitin ABC lyase I.
• The creation of a more thermostable
  chondroitinase ABC.