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Nucleotide Intermediates in the Biosynthesis of Polysaccharides.

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Nucleotide Intermediates in the Biosynthesis of Polysaccharides
I. Introduction and Sulfated Polysaccharides [*I
BY PROF. DR. JACK L. STROMINGER
DEPARTMENT OF PHARMACOLOGY. WASHINGTON UNIVERSITY SCHOOL OF MEDICINE,
ST. LOUIS, MISSOURI, USA.
In metabolism, nucleotides play a role not only as the precursors of nucleic acids. Many
low molecular weight compounds may be activated for subsequent biosynthesis by forming
anhydrides or esters with nucleotides. These nucleotide derivatives function as carriers of the
residues attached to them. Frequently the residues become chemically changed while attached to the nucleotide before transfer in a biosynthetic reaction. Following a general
survey of nucleotide-types, and their reactions, research into the biosynthesis of sulfared
polysaccharides is described.
I. Introduction
A little over 10years ago, Leloir and his collaborators [I]
discovered a co-factor for galactose utilization in yeast
isolated the compound, and showed that it was uridine
diphosphoglucose. Since these pioneering studies, it
has become increasingly apparent thet, in addition to
their role as precursors of nucleic acids, many dcidsoluble nucleotides have roles in other metabolic processes. There are four groups of acid-soluble nucleotides
related to the four principal ribonucleotides which occur
in ribonucleic acid (as well as several, recently discovered, which contain deoxyribonucleotides).The adenosine
nucleotides have been known for at least 25 years.
Soon after the isolation of uridine diphosphoglucose
(UDPG), guanosine and cytidine iiucleotides were also
discoverod as components of the acid-soluble fraction
of tissues. Each of these types of nucleotide has a special
metabolic function in addition to its role as a nucleic
acid precursor. By way of introduction, the general
types of compound for each nucleotide and their diverse
metabolic functions will be described [**I.
Adenosine Nucleotides
In the case of the adenosine nucleotides, there are, fist
of all, the oxidation-reduction intermediates, DPN,
TPN, and FAD. Each of these compounds, as well as
[*I This paper and a second one which is to appear later in this
journal are edited transcriptions of two lectures given at Freihurg
University in July, 1960, and as the McLaughlin Lectures at the
University of Texas in October, 1960. Work in the author’s
laboratory has been generously supported by the U.S. Public
Health Service (Grants NIAMD A-1158 and NIAID E-1902)
and by the U S . National Science Foundation (Grant G-7619),
to whom I am greatly indebted.
[**I Documentation of the first half of this paper would require
a bibliography of several hundred references. Therefore only a
limited number of references are given, but the sources of the
work described have been fully covered in several reviews [2-51,
including one by the author [6].
[ l ] R . Capurto, L. F. Leloir, C . E. Cardini, and A . C . Paladini, J.
biol. Chemistry 184, 333 (1950).
134
most of the others, can be considered as the acid
anhydride of a mononucleotide, i.e. a substituted phosphoric acid and another acid. For example, DPN is the
anhydride of adenylic acid and nicotinamide ribose
phospl-ate, and FAD is the anhydride of adenylic acid
and riboflavin phosphate. These compounds are synthesized primarily by reaction between a nucleoside triphosphate (ATP in the case of the adenosine nucleotides; and the acid which is to be activated (Equation 1).
XTP
+ acid
Z XMP-acid
+ PP
X
=
nucleoside
(1)
For example, a reaction between ATP and riboflavin
phosphate leads to formation of the anhydride, flavin
adenine dinucleotide (FAD), with the simulatenous
splitting-out of inorganic pyrophosphate. This mechanism for the synthesis of these acid anhydrides by means
of displacement of inorganic pyrophosphate from a
nucleoside triphosphate by another acid was first discovered by Kornberg during studies of DPN metabolism [2].
In addition to the oxidation-reduction intermediates, a
number of other types of compounds are activated as
derivatives of adenylic acid, e.g. amino-acids. In recent
years, adenyl amino-acids have been isolated and synthesized, and are believed to be intermediates in the biosynthesis of proteins. Another compound activated in
this way is inorganic sulfate. Adenyl sulfate is formed as
the product of the primary reaction with ATP (Equation
2). In this latter case, however, the equilibrium is
extremely unfavorable and in the living cell it is displaced by the phosphorylation of the adenylic acid
moiety (Equation 3). The product is “active sulfate,”
3’-phosphoadenosine-5‘-phosphosulfate
(PAPS). This
compound has a role in the biosynthesis of chondroitin
sulfates and of other naturally occurring sulfate esters [7] (Equation 4).
+ sulfate $ AMP-sulfate + PP
AMP-sulfate + ATP -+ PAPS + ADP
PAPS + acceptor + acceptor-sulfate + PAP
ATP
(2)
(3)
(4)
[2] A . Kornberg, Advances in Enzymol. 18, 191 (1957).
Angew. Chem. internat. Edit. / Vol. I (1962) 1 No. 3
Uridine Nucleotides
The second group of compounds is the uridine nucleotides [3,6]. These compounds are anhydrides of
uridylic acid and a sugar phosphate, ie.. UDP-sugar
compounds (Equation 5). A very large number of such
compounds are now known, and they serve many
important metabolic roles in transglycosylation reactions (Equation 6) and in transformations of one
sugar to another.
(5)
UTP
+ glucose-]-phosphate + UDP-glucose + PP
+ fructose + sucrose + UDP
(6) UDP-glucose
Guanosine Nucleotides
The third group of compounds is the guanosine nucleotides [4,6]. However, this group has not yet been
very extensively studied. Only recently has the isolation
of some new compounds stimulated further interest.
The earliest isolated was guanosine diphosphomannose
(GDP-mannose [8]). Its precise metabolic role is still
unknown, but it is presumably an intermediate in the
synthesis of mannose-containing polysaccharides. GDPD-Mannose is also the precursor of GDP-I--fucose[9].
is, therefore, synthesizedby
Fucose (6-deoxy-~-galactose)
living organisms in the form of its nucleotide derivative.
Fucose is avery important component of the blood-group
substances, as well as of many other types of polysaccharides. GDP-D-Mannose can also be transformed
to GDP-colitose [lo]. Colitose is a 3,6-dideoxy-hexose
(3,6-dideoxy-~-galactose),
found in the 0-endotoxin of
several gramnegative bacteria. For example, it occurs in
the 0-endotoxin of some pathogenic E. coli strains,
which are the causative agents of infantile diarrhea.
The mechanism involved in the synthesis of O-endotoxins and of their precursors is the subject of very
active investigation.
Cytidine Nucleotides
Finally, the compounds which are activated as derivatives of cytidylic acid, are, with one exception, alcohols [5, 61. One alcohol which is activated as a cytidine
nucleotide is choline (Equation 7). CDP-Choline is a
key intermediate in phospholipid synthesis (Equation
8) is]. It may be noted that in the synthetic reaction a
phospho-alcohol is transferred (Equation 8) and cytidine monophosphate (CMP) is the second product.
(7)
(8)
+ phosphocholine + CDP-choline + PP
CDP-choline + diglyceride + lecithin + CMP
CTP
[3] L . F . Leloir and C. E. Cardini in P. D . Boyer, H . Lardy, and
K . Myrback: The Enzymes. Academic Press, New York, 1960,
Volume 11, Chapter 2.
[4] M , F. Utter in P. D. Boyer, H . Lardy, and K. Myrback: The
Enzymes. Academic Press, New York, 1960,Volume 11, Chapter 4.
[5] E. P . Kennedy in P. D . Boyer, H . Lardy, and K. Myrback:
The Enzymes. Academic Press, New York, 1960, Volume 11,
Chapter 3.
I61 J . L. Strominger, Physiol. Rev. 40, 55 (1960).
[7] F. Lipmann, Science (Washington) 128, 575 (1958).
[8] E. Cabib and L . F. Leloir, J. biol. Chemistry 206, 779 (1954).
[9] V . Ginsburg, J. biol. Chemistry 235, 2196 (1960).
[lo] E. C. Heath, Biochim. biophysica Acta 39, 377 (1960).
Angew. Chem. internat. Edit. / Vol. I (1962) 1 No. 3
This is in contrast to synthetic reactions of uridine nucleotides where the sugar is transferred (Equation 6)
and UDP is the second product. Similarly, ethanolamine is activated in this way for the synthesis of
ethanolamine lipids. The synthesis of inositol lipids also
takes place through a mechanism involving a cytidine
nucleotide, although a somewhat more complicated
mechanism is used than in the synthesis of choline and
ethanolamine lipids [l 11. Two compounds which have
so far been isolated only from bacteria are CDPglycerol and CDP-ribitol [12]. They are, presumably,
intermediates in the biosynthesis of components of
bacterial cell walls and of other bacterial heteropolymers. CMP-N-Acetylneuraminic acid is a very unusual
nucleotide in this group 1133.
Deoxyribonucleotides
Similar derivatives of nucleotides found in deoxyribonucleic acid have been isolated recently although so
far this group of compounds is very limited. DeoxyCDP-choline and deoxy-CDP-ethanolamine were the
first of these to be obtained in a pure state [14, 151.
More recently, thymidine diphosphosugar compounds
have been isolated (e.g. TDP-L-rhamnose [16, 171,
TDP-D-fucose [IS, 191, TDP-6-deoxy-~-glucose[18, 191,
TDP-Cketo-6-deoxy-~-glucose
[19,20], and TDP-mannose [21]. The occurrence of nucleotide derivatives
which are so closely related chemically to deoxyribonucleic acid raises the question as to whether they are
related functionally in any way to the genetic material.
II. Reactions of the Nucleotides
A large number of small molecules are activated for
synthetic reactions as derivatives of a variety of nucleotides. Acids are activated as adenosine nucleotides,
whilst sugars (aldehydes) are activated as guanosine,
uridine or thymidine nucleotides and alcohols are
activated as cytidine or deoxycytidine nucleotides.
Formula I gives the structure of one of these nucleotides,
namely, uridine diphosphoacetylglucosamine[22]. It
[ I l l H . Paulus and E. P. Kennedy, J. biol. Chemistry 235, 1303
( 1960).
[I21 J . Baddiley, Proc. chem. SOC.(London) 177 (1959).
[I31 D . G. Comb and S . Roseman, 3. Amer. chem. SOC.80, 497
(1958).
[I41 Y. Sugino, J. Amer. chem. SOC.79, 5074 (1957).
[15] W . C. Schneider and J. Rorherham, J. biol. Chemistry 233,
948 (1958).
[I61 R. Okazaki, Biochem. biophysical Res. Commun. 1, 34
(1959).
1171 R . Okazaki, Biochim. biophysica Acta 44, 478 (1960).
[IS] J. L . Strominger and S. S. Scott, Biochim. biophysica Acta
35, 582 (1959).
[19] R . Okazaki, T . Okazaki, J. L . Strominger, and A . M .
Michelson, (submitted for publication).
1201 R. Okazaki, T . Okazaki, and J. L . Strominger, Fed. Proc. 20,
906 (1961).
[21] J. Baddiley and N . L . Blumson, Biochim. biophysica Acta 39,
376 (1960).
[22] E. Cabib, L. F. Leloir, and C . E. Cardini, J. biol. Chemistry
203, 1055 (1953)
135
is a precursor of acetylglucosamine-containing polysaccharides, such as hyaluronic acid. The acid anhydride
bond at (a) is formed in the reaction between the nucleoside triphosphate and the sugar phosphate, and the
bond at (b) is split in the synthetic reaction involving
the transfer of the sugar to a growing polysaccharide
chain. The sugar can be considered as a fragment,
activated by the nucleotide for some synthetic process.
(cf. Equation 6) and in recent years it has been shown
that glycogen is not synthesized directly from glucose-1phosphate but rather from UDP-glucose. UDP-Galactose is used in the synthesis of lactose and of cerebrosides in brain. UDP-Galacturonic acid undoubtedly
has many important metabolic roles which have not
yet been adequately described. Two congenital defects
in uridine nucleotide metabolism occur in man. One
of these is a defect in formation of UDP-galactose from
galactose-1-phosphate (galactosemia) and the other
one is a defect in the transfer of glucuronic acid form
UDP-glucuronic acid to bilirubin (congenital nonhemolytic bilirubinemia).
Many sugars are activated in this way, and in Diagram I,
the UDP-sugar compounds related to UDP-glucose are
(?
(a)
\
? - d l 0-C1
'0-
HC-C-C-C-CH20-P-0-PI!X
H
I
/A
0
"
ll
0
1
i
AH H
OH
I
I
I
1
,
I
C-C-C-C-CH,OH
H H
O H H
H
Glucuronide Synthesis
Glucuronide synthesis provides an excellent example
of a typical reaction cycle for one of these nucleotides
(Diagram 2). It is a mechanism by which many drugs
and other foreign organic compounds as well as normal
metabolites are conjugated prior to excretion. Firstly,
uridine triphosphate is formed from uridine diphosphate. Next, glucose-1-phosphate is activated by reaction with UTP. Then the sugar moiety of UDPglucose is modified, in this case by oxidation, to UDP-
N
,
=
C
'
'CH
k
&H
I
iC/
OH
I
shown. A primary reaction in this scheme is the reaction of glucose-1-phosphatewith uridine triphosphate
torgive UDP-glucose with the formation of inorganic
1
UDP-
\
1
UDP-
1
Ascorbic acid
'*..,
1
1 UDP-Galactose 1
dations
[UDP-Rhamnose]
Cerebrosides
<
7(other glycolipids?)
-
Lactose phosphate
A
Polysaccharides
-___.
1
'.x
Ascorbic acid
Diagram 1. Uridine Diphosphohexoses
UDP = Uridine diphosphate
UTP = Uridine triphosphate
PP = Pyrophosphate
G-1-P = Glucose-1-phosphate
Gal-I-P = Galactose-I-phosphate
GA-I-P = Glucuronic acid-I-phosphate
GalA-1-P = Galacturonic acid-]-phosphate
DPN = Diphosphopyridine nucleotide
(1) Enzymatic defect in congenital galactosemia
(2) Enzymatic defect in congenital non-hemolytic bilirubinemia
pyrophosphate (Equation 5). Once activated in the form
of its nucleotide derivative, the glucose moiety can be
modified in a number of ways. UDP-Glucuronic acid,
UDP-galacturonic acid, and UDP-galactose are formed
through transformation of UDP-glucose. Each of these
sugar-containing nucleotides can participate in a synthetic reaction. For example, UDP-glucuronic acid is
used in the synthesis of glucuronides. UDP-Glucose is
an intermediate in the biosynthesis of sucrose in plants
136
glucuronic acid. Finally, the modified sugar is transferred to an acceptor (e.g. bilirubin) with formation of a
glucuronide. The UDP which is formed as the second
product of this reaction can then cycle again. The
essential features of this cycle are: 1. phosphorylation,
2. activation of the sugar, 3. modification of the sugar
and finally, 4. transfer of the sugar to an acceptor in the
synthetic reaction.
To comment briefly on the reaction by which glucuronic acid
is formed from UDP-glucose (Equation 9) [23], it is one of
two examples of an oxidation which involves a four electron
(9)
UDP-glucose
+
2 D P N + -+
UDP-glucuronicacid
+
2DPNH
+ 2H+
transfer, and for that reason is of considerable interest. The
enzyme which catalyzes the oxidation of the amino alcohol,
[23] J. L. Strominger, E. S. Maxwell, J. Axelrod, and H . M.
Kalckar, J. biol. Chemistry 224, 79 (1957).
Angew. Chem. internat. Edit.
VoI. I (1962) No. 3
histidinol, to the amino-acid histidine [24], is the only other
known example of such an enzyme. All other known biological oxidations involve either one-or two-electron transfers.
Energy Generation
I
ATPK
PAD’
Glycogen
v
R-Glucuronide,
.UDP
1(b-
-1
UTP,
There are two defects in enzymes of this cycle which occur in
human beings. One of these is the inability of the species to
form ascorbic acid, which man shares with monkeys and
guinea pigs. The other defect is in the conversion of Lxylulose to xylitol, a benign condition known as congenital
pentosuria. The consequence of this defect is the excretion of
very large amounts of L-xylulose. The L-xylulose originates
from free glucuronic acid formed from the uridine nucleotide.
$3-1-P
Plant Uridine Nucleotides
ROH’
UDP-Glucuronic
acidK
UDPG
Plants also have mechanisms for activating sugars as
nucleotide derivatives. One of these mechanisms in
plants is the activation of the pentoses, xylose and
arabinose, as UDP-derivatives for the synthesis of
xylans and arabans, important constituents of many
plants (Diagram 4) 1251.
‘LPP
v
2 DPN+
,
2DPNH~
2H+
+
Balance:
ATP
+ G-1-P + 2 DPN+ + ROH
ADP
UDP
_3
+ 2 DPNH + 2 H+ + PP + R-Glucuronide
UDP-Glucuronic acid
Diagram 2. Cyclic Mechanism of Glucoronide Synthesis
ATP = Adenosine triphosphate
ADP = Adenosine diphosphate
UDP = Uridine triphosphate
G-I-P = Glucose-1-phosphate
PP = Pyrophosphate
UDPG = Uridine diphosphoglucose
DPN = Diphosphopyridine nucleotide
DPNH = Reduced diphosphopyridine nucleotide
II
UDP-Arabinose
.---+ Arabans
,? -co*
The Glucuronic Acid Cycle
I
In addition to serving as a donor of glucuronic acid for
the synthesis of glucuronides, UDP-glucuronic acid has
another important metabolic role as a precursor of free
glucuronic acid, both for ascorbic acid synthesis and
for a third cycle of glucose catabolism which occurs
in man as well as in other animals (Diagram 3). This
cycle is known both as the “uridine nucleotide shunt”
and as the “glucuronic acid pathway.”
Glucose
Pentose phosphate
D-Xylulose-5-P
ATP
T
_____f
pathway
-1
G-6-P
+G-l-PK
UDP-Acetylamino Sugars
Finally, the acetylamino sugars comprise a third group
of sugars which are activated as uridine nucleotides
(Diagram 5). An important reaction is that between
UTP t
-
fl
U D P 6 -PP
i2DPN
‘1
Xylitol
TPNH
Diagram 4. Uridine Diphosphopentoses.
Xyl-I-P = Xylose-1-phosphate
Arab-1-P = Arabinose-1-phosphate
See also keys to Diagrams 1 and 2
>(
0-Xylulose
DPN
I
UDP-Galacturonic acid
@
H20
UMP
,/
UDPG:ucuronic acid
\ ROH
+ GA-1-P
HzO
R-Glucuronide
1
L-Xylulose
+U
,/ HzO
Glucuronic acid
n,
/I TPNH
I,I
3-KetoDPN
-[r-gu,onic
acid] t L-Gulonic acid
11IL
L-Gulono-ylactone
=
I @
UTP and acetylglucosamine-1-phosphate, leading to
the formation of UDP-acetylglucosamine, which can
then be modified in a number of ways. First of all,
it can be transformed to UDP-acetylgalactosamine.
Further reactions, which are not yet understood, lead to
the synthesis of UDP-acetylgalactosamine-4-sulfateand
UDP-acetylglucosamine-6-phosphate[*]. These unusual
J
Degradation products t %-Ascorbicacid
Diagram 3. The Glucuronic Acid Cycle
G-6-P = Glucose-6-phosphate
UMP = Uridine monophosphate
TPNH = Reduced triphosphopyridine nucleotide
See also keys to Diagrams 1 and 2.
[24] E. Adums, J. biol. Chemistry 217, 325 (1955).
Angew. Chem. internat. Edit. 1 Vol. I (1962)I No. 3
1251 W . Z . Hassid, E. F. Neufeld, and D. S . FeingoId, Proc. nat.
Acad. Sci. U.S.A. 45, 905 (1959).
[ * ] It has been found recently that UDP-acetylglucosamine-6phosphate [26] is a degradation product of the nucleotide actually present in hen oviduct. The structure of this compound,
UDP-acetylglucosamine-6-phospho-I-D-galactose was deduced
independently in two laboratories 127, 281. The galactose moiety
in the nucleotide is extremely labile.
137
compounds have been isolated from hen oviduct.
Substances of this type, as well as UDP-acetylglucosamine, are presumably precursors of various polysaccharides, including the chondroitin sulfates and hyaluronic
acid. The group of compounds which appear at the
bottom of Diagram 5 are precursors of bacterial cell
walls.
hens [26]. The acetylgalactosamine-4-s~dfate
moiety,
activated in the form of the uridine nucleotide, is presumably a precursor of some sulfated polysaccharide.
In order to consider a possible function of this compound, it is necessary to appreciate the function of the
oviduct. The yolk enters from the abdominal cavity
through the infundibulum and passes first into a region
about 20 cm. in length where the albumin is secfeted.
Polysaccharides
m
11
-..,) UDP-Acetylglucosamine-4-sulfate
UDP-Acetylgalactosamine
U
:
IT
x
l
1
NAcG-l-P
t
PP
UDP-Acetylglucosamiue
1
Chitin,
hyaluronic
acid
'
,
7
UDP-Acetylglucosamine-Cphosphate
/a
1 .:'
1 L\
UDP-Acetylglucosamine
pyruvate
and
intermedicates in
bacterial cell wall
synthesis
(cf. Fig. 3)
+
UDP Acetylmannosamine
Polymer:containing
acetylneuraminic acid
(e.g. gangliosides,
coliminic acid)
Diagram 5. Uridine Diphosphoacetylamiuo Sugars
NAcG: 1-P = N-Ace tylgIucosamine-1-phosphate
PP = Pyrophosphate
Sulfated,
phosphorylated
or branched
polysaccharides
This is by far the largest section of the oviduct. The egg
then passes into a very narrow area about two centimeters in length, the isthmus, in which the inner shell
membranes are secreted around the albumin. These
membranes are secreted over a period of abour an hour.
Their structures are still not known, but they do contain
a sulfated mucopolysaccharide of some kind. Calcification takes place in the third region of the oviduct
(uterus). The terminal region (vagina) is the region where
the smooth outer surface of the egg (cuticle) is secreted,
immediately before the hen lays the egg.
Table 1. Sulfate Metabolism in the Oviduct of the Laying Hen 1291
Function of
Segment
Segment
I
Infundibulum
Secreting
Repion
1 7I
1 II I
I\
Vagina
III. The Structure and Biosynthesis of Chondroitin
Sulfates
With this background, current knowledge of the mechanism of synthesis of the chondroitin sulfates may be
discussed and the role of uridine, and other, nucleotides
in the biosynthesis of polysaccharides examined. Some
interesting data relating to the polysaccharide structure
was obtained from studies of the biosynthesis of chondroitin sulfates.
I
Albumin
Formation
UDPGalNAc-S
in Oviduct
3SSO;-
in
Oviduct
0
0
0
3+
4+
(membranes)
4+
Calcification
(including shell
matrix formation:
0
?
Cuticle
Formation
4-t
2+
(cuticle)
Formation
In order to find out where sulfate metabolism occurred
in this complicated route ,3sS-inorganicsulfate was given
to living hens. It was taken up only in two discrete
regions of the oviduct, the isthmus and the vagina, in
each of which shell membranes are synthesized (see
Table 1). In both of these regions, 35s was located in the
glandular epithelium. Eggs laid by these hens were also
examined; both the inner shell membranes and the
cuticle were radioactive [29].
The distribution of the sulfated uridine nucleotide in the
segments of oviduct was examined next. This nucleotide
The Nature and Function of the Hen Oviduct
[26] J. L. Strominger, Biochim. biophysica Acta 17, 283 (1955).
These studies began with the isolation of UDP-acetylgalactosamine-4-sulfate from the oviduct of laying
[27] S. Suzuki, submitted for publication.
[28] 0.Gabriel and C . Ashwell, submitted for publication.
[29] J . L. Strominger and S. Suzuki, unpublished observations.
138
Airgew. Chem. internal. Edit. 1 VoI. I (1962) /
NO.3
was found only in the isthmus. Its concentration in this
region is about 5 micromoles/gram, an extremely high
concentration for a nucleotide in a tissue. When a paper
chromatogram was prepared from a hot water extract
of isthmus, the sulfated uridine nucleotide was the only
detectable ultraviolet-absorbing compound. These data
suggested that isthmus was an excellent tissue with which
to study the synthesis of sulfated mucopolysaccharides.
The Sulfation of Polysaccharides
At about this time Robbins and Liprnann[30] had
isolated phosphoadenosine phosphosulfate (PAPS). It
seemed plausible that PAPS might be the sulfate donor
for the synthesis of UDP-acetylgalactosamine-sulfate
and that the reaction might be a direct sulfation of UDPacetylgalactosamine. 3%-Labeled phosphoadenosine
phosphosulfate was prepared using enzymes isolated
from isthmus. Incorporation of 3 5 s from PAP35S into
chondroitin sulfate was studied in the belief that the
reaction sequence would lead through the sulfated
uridine nucleotide. When PAP35S was incubated with
enzyme from isthmus, the major reaction was hydrolysis
of PAPS to inorganic sulfate. If, however, an acceptor,
such as chondroitin sulfate A, was also added, a new
sulfated product was formed which remained at the
origin on a paper chromatogram (Figure 1 ) and which
was then recovered and shown to be radioactive
chondroitin sulfate A (Table 2) [31].
Table 2. Synthesis of Sulfated Mucopolysaccharides from P A P W [31].
In these experiments 3 5 s sulfate was transferred from P A P W of high
specific activity to various acceptors, catalyzed by an enzyme preparation
from the isthmus of hen oviduct [311. Data are recorded as c.p.m. incorporated into polysaccharide acceptor under the conditions employed
In experiment 1, omission and substitution of various components of
the system was studied. In experiment 2, the relative rates of sulfation
of various acceptors was examined,
Radio-activity
of the
polysaccharide
[c.p.m.l
Experiment 1
System with chondroitin sulfate A
as acceptor
I. Complete
2. Minus chondroitin sulfate A
(added after incubation)
3. Minus P A P W (added after incubation)
4. Boiled enzyme control
S. AP3% substituted for PAP%
3320
Experiment 2
Various acceptors
Chondroitin sulfate A
Chondroitin sulfate E
Chondroitin sulfate C
Heparitin sulfate
Heparin, hyaluronic acid, glycogen or
charonin sulfate
1750
1330
1060
1000
Rf
0.87
-p-nitrophenyl sulfate
-5’-AMP
0.81
0.79
-tyramine sulfate
-ATP and APS
0.52
-PAPS
0.42
-so4
0.35
-UDP-GalNAc
0.31
-@
and UDP-GalNAc-S
-Chondroitin sulfate A
I
A
1
B
I
C
I
D
0.49
0.20
0.14
-a
0.00
1
E
Fig. 1. Radio-autogram of a Chromatogram of lncubation Mixtures
Containing PAPJsS and an Enzyme Preparation from the Isthmus of
Hen Oviduct [31].
A: Inorganic sulfate
B: PAP3sS and an enzyme preparation heated before incubation
C: PAP3sS and enzyme preparation
D: As C, but with SO Fg chondroitin sulfate A that had been partially
hydrolyzed with hyaluronidase. The substance with Rf = 0 is the polymer,
the spots between Rf = 0 and 0.14 correspond to sulfated oligosaccharides. If the polymer is not treated with hyaluronidase before incubation,
only the spot at Rf = 0 is found.
E: Chromatogram with standard substances
a and p are unidentified radioactive products. @ is not UDP-GalNAc-S
although it has the same mobility in this solvent.
sulfate, and, finally, a chondroitin sulfate which had
been isolated in Japan from shark cartilage and which
was believed to be identical to chondroitin sulfate C
(Figure 2). However, the apparent K, and Vmax for
this latter compound were different from those for
chondroitin sulfate C. These data suggested that the
compound might not be identical with chondroitin
sulfate C. Other data (see below) prove that it is, in fact,
a novel form of chondroitin sulfate.
30
25
30
100
Radio-activity
of the
polysaccharide
[c.p.m.]
-3’-AMP
The Course of the Sulfation Reaction
In considering the mechanism by which sulfate was
incorporated into the polysaccharides in this system, the
idea that the sulfated uridine nucleotide (UDP-GalNAcS) was an intermediate in the incorporation, and that
the polysaccharide served as an acceptor for acetylgalactosamine-sulfate (transferred from the uridine
nucleotide), was attractive (Equations 10 and 11). HowPAPS
UDP-GalNAc d UDP-GalNAc-S
40 to 50
.
(10)
UDP-GalNAc-S
A number of polysaccharides would serve as acceptors
in this system, viz. chondroitin, chondroitin sulfate A,
chondroitin sulfate B, chcndroitin sulfate C, heparitin
[30] P. W. Rubbins and F. Lipmann, J. biol. Chemistry 233, 681,
686 ( I 958).
[3 I J S. Suzuki and J . L. Sfruminger,J. biol. Chemistry 235, 257,
267, 274 (1960). See also [32].
Angew. Chem. internat. Edit.
Vul. I (1962) No. 3
Polysaccharide
Polysaccharide-GalNAc-S
(1 1)
ever, a second possibility had to be considered, viz. that
the uridine nucleotide was not an intermediate, but that
the polysaccharide itself was the primary acceptor in the
trans-sulfation (Equation 12).
PAPS
Polysaccharide
Polysaccharide-S
(12)
139
I
^^^^
I
/ o -
7
sulfate were also both sulfated, with the formation of
acetylgalactosamine monosulfate or acetylgalactosamine disulfate, respectively, as the products (Figure 3).
m
0
20
40
LA175.21
Fig. 2. Relationship between the Concentration of Various Acceptors
and Reaction Velocity [311.
Curve 1 : Chondroitin sulfate C
Curve 2: Chondroitin from bovine cornea
Curve 3: Chondroitin sulfate A
Curve 4: Chondroitin sulfate B
Curve 5 : Chondroitin prepared by chemical desulfation of chondroitin
[sulfate
Curve 6: Heparitin sulfate
Curve 7: Chondroitin sulfate from shark cartilage
Curve 8: Glycogen, heparin, keratosulfate, hyaluronic acid or charonin
sulfate
Ordinate: Radio-activity of polysaccharides [c.p.m.l
Abscissa: pg. Acceptor per 50 pl. incubation mixture
It was impossible to study this problem using the large
polymers as acceptors because the simple addition of
3%-sulfate to these high molecular weight substances
could not be distinguished from addition of acetylgalactosamine-3%-sulfate. The sulfation of low molecular weight oligosaccharides derived from chondroitin
and from chondroitin sulfate A by enzymatic hydrolysis
was, there fore^ investigated. In this case, it was relatively
easy to distinguish the possible products of sulfation by
paper chromatography. For example, if the tetrasaccharide containing no sulfate residues was used as
an acceptor the possible products, tetrasaccharide
monosulfate or pentasaccharide monosulfate, could
be easily distinguished. To prepare the sulfated products, enzyme and PAP35S were incubated with various oligosaccharide acceptors. The incubation mixtures were then chromatographed, and in each case
a new radioactive product was formed which had a
slower mobility than the acceptor itself. This radioactive
product was eluted from the chromatogram and its
mobility determined during paper chromatography and
paper electrophoresis, together with authentic standards. In each case, it was found that the sulfation reaction was a simple transfer of sulfate from the donor
to oligosaccharide (corresponding to Equation 12).
Acetylgalactosamine and acetylgalactosamine mono140
Fig. 3. Sulfation of Mono- and Tetrasaccharides 1311.
Positions of the sulfated compounds are derived from paper chromatography (abscissa) and paper electrophoresis (ordinate). The chromatograms were developed with butanol/acetic acidiwater.
Open circles: Added as acceptor
Full circles: Radioactive product
Arrows indicate the relationship between acceptors and products.
A : N-Acetylgalactosamine
B : N-Acetylgalactosamine monosulfate
C: N-Acetylgalactosamine disulfate
D : Tetrasaccharide
E : Tetrasaccharide monosulfate
F: Tetrasaccharide disulfate
G: Tetrasaccharide trisulfate
Ordinate: Distance covered during electrophoresis Icm.1
Abscissa: Rglucuronic acid
The relative dependence of the sulfation velocity on the
chain length is indicated in Figure 4.
70000-
I
O A
I
I
601xIo -
50000 -
lA775.41
Di
Tetra
Hexa
Fig. 4. Relationship between Chain Length of the Acceptor and the
Velocity of Sulfate Transfer (311
-0-0- sulfate-free acceptor
-x-x- sulfated acceptor
A: Cbondroitin
B: Chondroitin sulfate A
Ordinate: c.p.m. incorporated.
Angew. Chem. internat. Edit. / Vol. I (1962) 1 No. 3
Although most of these compounds were sulfated at
rates which were relatively slow, the trisaccharide
(GalNAc-GA-GalNAc) and the pentasaccharide
(GalNAc-GA-GalNAc-GA-GalNAc)[*] were sulfated
at a rate comparable to that of the polymer itself. This
may be an important point. The even-numbered oligosaccharides all contained glucuronic acid on the nonreducing end of the oligosaccharide chain. The oddnumbered oligosaccharides were prepared from the
even-numbered compounds by removal of the terminal
glucuronic acid residue with a p-glucuronidase, yielding
oligosaccharides with an acetylgalactosamineresidue on
the non-reducing end. These odd-numberedoligosaccharides were extraordinarily efficient as acceptors in the
sulfation system. It is possible, therefore, that at least
urement of both periodate consumption and formaldehyde liberation distinguishes between all of the possible
isomeric acetylgalactosamine sulfates. It has been established that the acetylgalactosamine monosulfate derived
from the nucleotide was acetylgalactosamine-4-sulfate
while the acetylgalactosamine monosulfate prepared by
chemical sulfation is acetylgalactosamine-6-sulfate.The
acetylgalactosamine monosulfates were then isolated
from several polysaccharides, and by comparison with
the now characterized materials, it has been established
that the compound from chondroitin sulfate A or B was
the 4-sulfate, while that from C was the 6-sulfate,
thereby confirming by direct chemical isolation what
other investigators had previously concluded from
other studies (Table 3).
Table 3. Identification of Acetylgalactosamine Monosulfates [331
1. GalNAc-4-S from uridine nucleotide
2. GalNAc-64, chemically synthesized
3. GalNAc-S from Chondroitin sulfate A
4. GalNAc-S from Chondroitin sulfate B
5. GalNAc-S from Chondroitin sulfate D
Rfin
Isobutyric
acidiNH4OH
Moles of
HCHO
formed/mole['I
0.52
0.92
0.05
0.62
[+I
0.75
['I
0.44
0.53
(trace at 0.44)
0.52
0.44
0.03
ESES
in
Morgan-Elson
reaction
Infrared peaks[**]
[cm-11
200
10 000
1 800 ('1
695; 860; 890; 920
775; 820; 992
695; 860; 890; 920
1 120 [+I
10 000
695; 860;890; 920
780; 815; 992
[*I With periodate oxidation at 25OC. for 20 hours. GalNAc-4-S consumed 1 mole of periodate while GalNAc-6-S
consumed 4 moles.
['*I Peaks in the infrared spectra between 660 and 1000 cm-1.
[+I These values suggest that these samples (predominantly GalNAc-4-S)
one mechanism for the introduction of sulfate into the
polysaccharides is sulfation of an acetylgalactosamine
residue on the non-reducing end of a growing polysaccharide chain. However, the role of the sulfated uridine nucleotide still remains to be elucidated, and there
may be several mechanisms for the introduction of sugars and of sulfate into polysaccharide chains.
may contain 10-15 % of GalNAc-6-S.
Two interesting aspects of the structure of chondroitin
sulfates arose from these enzymatic studies. Acetylgalactosamine itself is a sulfate acceptor. The product,
an acetylgalactosamine monosulfate, was electrophoretically identical to acetylgalactosamine monosulfate derived from the uridine nucleotide by hydrolysis,
as well as to acetylgalactosamine sulfate prepared by
chemical sulfation of acetylgalactosamine with chlorosulfonic acid. However, examination of these three
acetylgalactosamine monosulfates by chromatography
indicated that the compound from the uridine nucleotide
had a greater mobility than the compound which had
been prepared chemically. The enzymatically synthesized product was identical to that derived from the
uridine nucleotide.
The structure of these acetylgalactosaminemonosulfates
has been examined [33]. Periodate oxidation with meas-
The second aspect of this investigation of the mechanism
of chondroitin sulfate synthesis was the discovery of a
new form of chondroitin sulfate, which was called
chondroitin sulfate D, a compound first isolated by
Egami and co-workers in Japan from shark cartilage
[34]. As already mentioned, this compound was not
identical to chondroitin sulfate C as an acceptor for the
sulfotransferase, and the compound had, furthermore,
been found to contain about 2 2 % sulfate (i.e. 1.3
sulfate residues per acetylgalactosamine residue). In
order to investigate its structure, Suzuki [35] employed
an enzyme from Proteus vulgaris [36]. This enzyme
catalyzes an essentially quantitative degradation of
chondroitin sulfates to disaccharides. The reaction is an
elimination reaction, rather than a hydrolysis, so that
the product is a A4-unsaturated disaccharide [36]. When
the degradation products of various chondroitin sulfate
preparations were separated by paper chromatography,
chondroitin sulfate D yielded an unexpected product
which was not formed on degradation of either chondroitin sulfate A or of chondroitin sulfate C. A small
amount of a similar product was also formed by
degradation of a preparation of chondroitin sulfate B.
However, these two compounds could be separated by
paper chromatography, and are believed to be isomers.
On analysis, each of these compounds contained two
sulfate residues per disaccharide unit. This structure is
I'[ GalNAc = N-Acetylgalactosamine;GA = Glucuronic acid.
[32]S. Suzuki, R . H . Threnn, and J. L. Strominger, Biochim.
biophysica Acta 50, 169 (1961).
[33]S.Suzuki and J . L. Strominger, J. biol. Chemistry 235, 2768
(1960).
I341 T . Soda, F. Egami, and T . Horigome, J. chem. SOC. (Japan)
16,43 (1940).
[35]S . Suzuki, J. biol. Chemistry 235, 3580 (1960).
[36]K. S.Dodgson and A . G. Lloyd, Biochem. J. 68,88 (1958).
The Structure of the Chondroitin Sulfates
Angew. Chem. internat. Edit. Vol. I (1962) I No. 3
141
in line with the high electrophoretic mobility of the
disaccharide (Figure 5). Various data suggest that one
sulfate residue of both of these compounds is located on
the 2- or 3-position of the uronic acid moiety. For
example, the periodate consumptions of the disulfated
disaccharides are very much reduced, indicating that
the glucuronic acid moieties are substituted in some
2
la77sTm
I
l
l
A
B
C
mzm
Fig. 5. Relative Mobilities of Unsaturated Disaccharide from Chondroitin sulfate B and Chondroitin sulfate D [36]
A: Unsaturated disaccharide disulfate from chondroitin sulfate D
B: Unsaturated disaccharide-6-monosulfate
C: Sulfate-free unsaturated disaccharide
D : Unsaturated disaccharide disulfate from chondroitin sulfate B
manner, presumably by sulfate (Figure 6). The other
sulfate residue is on the acetylgalactosamine. The proposed structures of unsaturated disulfated disaccharides
from shark cartilage chondroitin sulfate and from a
chondroitin sulfate B preparation [36] are given in
Formulae I1 and 111.
The mechanism by which uridine nucleotides and other
nucleotides participate in the biosynthesis of sulfated
mucopolysaccharides is a very complicated problem.
These compounds can contain at least four types of
sulfate residues (4-0-, 6-0- or N-sulfate on the amino
sugar and sulfate on the uronic acid) as well as several
different sugars. Several types of mechanisms undoubtedly exist both for introduction of sulfate and for
introduction of sugars into the polysaccharide chains.
Undoubtedly there are more surprises to come, both
in terms of biosynthetic mechanisms and in terms of
structure. With the discovery of a number of nucleotide
intermediates, it may be hoped that more rapid progress
142
6
10
22 40-55
14
Fig. 6. Rate of Periodate Consumption of Unsaturated Disaccharides
1361
Curve A: Sulfate-free unsaturated disaccharide
Curve B: Unsaturated disaccharid-4-sulfate
Curve C: Unsaturated disaccharide-6-sulfate
Curve D: Unsaturated disaccharide monosulfate from chondroitin
sulfate D
Curve E: Unsaturated disaccharide disulfate from chondroitin sulfate B
Curve F: Unsaturated disaccharide disulfate from chondroitin sulfate D
Note the low periodate consumption of the disulfates (Curves E and F)
compared with the periodate consumption of the monosulfates (Curves
B to D) and the sulfate-free disaccharide (Curve A).
Ordinate: Moles IO;/mole disaccharide
Abscissa: Time [hours]
r--,OH
'
HC--
C-
1
I
I
H C - p
I
I
HOCH
I
I
i
HC
I
COOH
CHpOSOzH
------1
OH
I
COOH
CHzOH
111
can be made in the study of the biosynthesis of the
chondroitin sulfates as well as of other biologically
important heteropolymeric substances.
Received September 29, 1961
Angew. Chem. internat. Edit.
[A 175/6 IE]
Vol. I (1962)1 No.3
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