Method for producing sulfated lactosamine oligosaccharide

Disclosed is a method for producing a sulfated lactosamine oligosaccharide, comprising the step of allowing a sulfotransferase to act on a lactosamine oligosaccharide, the sulfotransferase transferring sulfate group to hydroxyl group at C-6 position of galactose residue in the lactosamine oligosaccharide.

BACKGROUND OF THE INVENTION 
The present invention relates to a novel method for producing a sulfated 
lactosamine oligosaccharide. In particular, the present invention relates 
to a method for producing a sulfated lactosamine oligosaccharide from a 
lactosamine oligosaccharide based on the use of an enzyme reaction. 
Sugar chains composed of lactosamine backbones serve as the ligand sugar 
chain which has the ability to bind cell adhesion molecules belonging to 
the selectin family, and thus they express important physiological 
activities in vivo. It has been clarified that expression of such sugar 
chains on cell surfaces participates in specific adhesion between 
heterogeneous cells. Functional activating agents and suppressing agents 
based on the utilization of these sugar chains are expected to be useful 
drugs. Namely, it is believed that these sugar chains include their 
principal compound of sialyl Lewis.sup.X (SLe.sup.X) and its analogs with 
which the selectin molecule is blocked to inhibit adhesion between 
lymphocyte and endothelial cell, and thus they are useful to mitigate 
various diseases associated with inflammation. In addition, it is also 
believed that these sugar chains participate in adhesion to endothelial 
cells upon hematogenous metastasis of tumor cells, and they participate in 
adhesion to target cells upon infection with microorganisms. Therefore, 
these sugar chains are regarded to be important as materials to develop 
pharmaceuticals for preventing such diseases. 
On the other hand, one of the ligands for L-selectin (expressed on 
lymphocyte) which participates in homing of lymphocyte and rolling of 
leukocyte caused at an initial stage of inflammation is considered to be 
"GlyCAM-1 (highly glycosylated cell adhesion molecule-1, 
glycosylation-dependent cell adhesion molecule-1; expressed on blood 
vessel endothelium)", and the sugar chain occupies not less than 70% of 
GlyCAM-1. Recently, GlyCAM-1 attracts great attention together with a 
basic backbone of the sugar chain (NeuAc-Gal(6S)--(Fuc-)GlcNAc--R, wherein 
NeuAc represents an N-acetylneuraminic acid residue, Gal represents a 
galactose residue, (6S) indicates that hydroxyl group at C-6 position is 
sulfated, Fuc represents a fucose residue, GlcNAc represents an 
N-acetylglucosamine residue,--represents a glycoside linkage, and R 
represents a hydrogen atom or a sugar chain: this basic backbone is 
hereinafter referred to as "basic backbone of GlyCAM-1 sugar chain", if 
necessary). 
GlyCAM-1 and the basic backbone of its sugar chain are expected to play 
important roles in development of novel pharmaceuticals and in studies on 
physiological activities in vivo. It is desired to stably supply these 
substances. However, at present, they can be obtained only from animals 
which express GlyCAM-1. Therefore, obtainable amounts are minute. It is 
difficult to produce GlyCAM-1 and the basic backbone of its sugar chain by 
means of organic synthesis. However, no method has been known for 
producing a sulfated lactosamine oligosaccharide which can be converted 
into the basic backbone of the GlyCAM-1 sugar chain. 
The present invention has been made taking the aforementioned viewpoints 
into consideration, an object of which is to provide a novel method for 
producing a sulfated lactosamine oligosaccharide in order to obtain the 
basic backbone of the GlyCAM-1 sugar chain. 
SUMMARY OF THE INVENTION 
As a result of diligent studies by the present inventors in order to 
achieve the object described above, it has been found that a sulfated 
lactosamine oligosaccharide can be obtained by allowing a sulfotransferase 
to act on a lactosamine oligosaccharide, the sulfotransferase transferring 
sulfate group to hydroxyl group at C-6 position of galactose residue in 
the lactosamine oligosaccharide. Thus the present invention has been 
completed. 
Namely, the present invention provides a method for producing a sulfated 
lactosamine oligosaccharide, comprising the step of allowing a 
sulfotransferase to act on a lactosamine oligosaccharide, the 
sulfotransferase transferring sulfate group to hydroxyl group at C-6 
position of galactose residue in the lactosamine oligosaccharide. 
The term "lactosamine" herein include lactosamine (a substance in which a 
galactose residue is bound to a glucosamine residue through a glycoside 
linkage; represented by Gal--GlcN), as well as N-acetyllactosamine (a 
substance in which a galactose residue is bound to an N-acetylglucosamine 
residue through a glycoside linkage; represented by Gal--GlcNAc). In the 
formulas described herein, Gal represents a galactose residue, GlcNAc 
represents an N-acetylglucosamine residue, GlcN represents a glucosamine 
residue, SA represents a sialic acid residue, NeuAc represents an 
N-acetylneuraminic acid residue, Fuc represents a fucose residue, (6S) 
indicates that hydroxyl group at hydroxyl group at C-6 position is 
sulfated, and--represents a glycoside linkage. 
The term "lactosamine" herein includes lactosamine containing a sialic acid 
residue and/or a fucose residue. The term "lactosamine oligosaccharide" 
herein refers to an oligosaccharide containing at least one lactosamine, 
which includes, for example, lactosamine itself, oligosaccharides 
containing at least one lactosamine, and oligosaccharides comprising a 
basic backbone of a repeating structure composed of lactosamines. The term 
"lactosamine oligosaccharide" includes those containing a sulfated 
glucosamine residue (or a sulfated N-acetylglucosamine residue) and/or a 
sulfated galactose residue provided that they are lactosamine 
oligosaccharides containing at least one lactosamine having a galactose 
residue which is not sulfated at its hydroxyl group at C-6 position. The 
term "sulfated lactosamine oligosaccharide" herein means a substance in 
which sulfate group is added to a part or all of hydroxyl groups at C-6 
positions of non-sulfated galactose residues in the lactosamine 
oligosaccharide. The description of only "lactosamine" herein means 
lactosamine itself, as well as a lactosamine structure (backbone) in the 
lactosamine oligosaccharide. 
According to the present invention, a sulfated lactosamine oligosaccharide 
can be produced from a lactosamine oligosaccharide. The sulfated 
lactosamine oligosaccharide obtained by the method of the present 
invention is expected to be utilized as an intermediate to obtain the 
basic backbone of GlyCAM-1 sugar chain. GlyCAM-1 and the basic backbone of 
GlyCAM-1 sugar chain can be utilized as anti-inflammatory agents.

DETAILED DESCRIPTION OF THE INVENTION 
The sulfotransferase to be used for the method of the present invention is 
not specifically limited provided that the sulfotransferase transfers 
sulfate group to hydroxyl group at C-6 position of galactose residue in 
the lactosamine oligosaccharide. However, it is preferable to use a 
sulfotransferase transferring sulfate group to hydroxyl group at C-6 
position of galactose residue of glycosaminoglycan. It is more preferable 
to use a sulfotransferase transferring sulfate group to hydroxyl group at 
C-6 position of galactose residue of keratan sulfate. It is especially 
preferable to use a sulfotransferase purified by the present inventors 
from a culture supernatant of chick chondrocyte cultivated in a serum-free 
medium (Habuchi, O., Matsui, Y., Kotoya, Y., Aoyama, Y., Yasuda, Y., and 
Noda, M. (1993), J. Biol. Chem., 268, 21968-21974). The especially 
preferable sulfotransferase (hereinafter referred to as "chondroitin 
6-sulfotransferase" or "C6ST", if necessary) has the following physical 
and chemical properties: 
(1) action: 
The sulfotransferase transfers sulfate group from a sulfate group donor to 
hydroxyl group at C-6 position of N-acetylgalactosamine residue or 
galactose residue of glycosaminoglycan. 
(2) substrate specificity: 
The sulfotransferase transfers sulfate group to chondroitin, chondroitin 
sulfate A, chondroitin sulfate C, and keratan sulfate, but sulfate group 
is not substantially transferred to chondroitin sulfate E, dermatan 
sulfate, and heparan sulfate. Preferably, the chondroitin originates from 
squid skin, the chondroitin sulfate A originates from whale cartilage, the 
chondroitin sulfate C originates from shark cartilage, and the keratan 
sulfate originates from bovine cornea. Preferably, the chondroitin sulfate 
E originates from squid cartilage, the dermatan sulfate originates from 
pig skin, and the heparan sulfate originates from bovine kidney. C6ST also 
transfers sulfate group to chondroitin sulfate originating from chick 
embryo cartilage. 
(3) optimum reaction pH: 
The sulfotransferase has an optimum reaction pH in the vicinity of 6.4. 
(4) activation: 
The activity of the sulfotransferase is increased by protamine or 
MnCl.sub.2. 
(5) molecular weight: 
The sulfotransferase has a molecular weight of about 75 kilodaltons as 
estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis 
under a reduced condition. 
C6ST described above can be purified from cultured cells which express 
C6ST, such as chondrocyte, by combining methods for ordinarily purifying 
proteins and methods for ordinarily purifying sulfotransferases. 
Specifically, purification is preferably performed in accordance with a 
method described in J. Biol. Chem., 268, (29), 21968-21974 (1993). Namely, 
substantially homogeneous C6ST can be obtained, for example, from a 
culture supernatant of chick embryo chondrocyte cultivated with a 
serum-free medium by means of affinity chromatography based on the use of 
Heparin-Sepharose CL-6B (commercially available from Pharmacia LKB 
Biotechnology), wheat germ agglutinin-agarose (commercially available from 
Seikagaku Corporation), and 3',5'-ADP-agarose (commercially available from 
Sigma). 
The sulfotransferase can be also obtained by cloning a gene of the 
sulfotransferase usable in the present invention, introducing the cloned 
gene into an appropriate host, and expressing the gene in accordance with 
a known method. For example, DNA which codes for the sulfotransferase is 
isolated from a DNA library of an organism having the sulfotransferase 
usable in the present invention, by using an index of the sulfate group 
transfer activity specific to the hydroxyl group at C-6 position of 
galactose residue in the lactosamine oligosaccharide or glycosaminoglycan 
(preferably keratan sulfate). The isolated DNA is inserted into a vector 
by means of gene recombination, and the vector is then introduced into a 
host cell to perform expression therein. Thus the sulfotransferase can be 
obtained. Alternatively, cloning can be performed by preparing and using 
an antibody specific to the sulfotransferase. Alternatively, an N-terminal 
amino acid sequence of the sulfotransferase may be determined to perform 
cloning by using a probe of DNA having a nucleotide sequence deduced from 
the determined amino acid sequence. The expressed enzyme can be obtained 
in accordance with ordinary methods for enzyme extraction and 
purification. The extraction method specifically includes, for example, 
extraction based on the use of cell disruption, such as homogenization, 
ultrasonic treatment, osmotic shock, and freeze and thawing, extraction 
based on the use of a surfactant, and treating operations based on the use 
of a combination of the foregoing. The purification method specifically 
includes, for example, salting out based on the use of ammonium sulfate or 
sodium sulfate, centrifugation, dialysis, ultrafiltration, adsorption 
chromatography, ion exchange chromatography, hydrophobic chromatography, 
reverse phase chromatography, gel filtration, gel permeation 
chromatography, affinity chromatography, electrophoresis, and treatment 
operations based on the use of a combination of the foregoing. 
A method for measuring the sulfotransferase activity and a method for 
inspecting the position of sulfate group transfer will be described in 
detail in Examples. 
The lactosamine oligosaccharide usable in the method of the present 
invention is an oligosaccharide containing at least one lactosamine, such 
as lactosamine, an oligosaccharide containing at least one lactosamine, 
and an oligosaccharide comprising a basic backbone composed of a repeating 
structure of lactosamines. Among them, lactosamine itself, or the 
oligosaccharide comprising the basic backbone composed of the repeating 
structure of lactosamines is preferred. The oligosaccharide comprising the 
basic backbone composed of the repeating structure of lactosamines 
preferably comprises 2 to 10 units, more preferably 2 to 7 units, and 
especially preferably 2 units of the lactosamine structures. 
In the case of the oligosaccharide containing at least one lactosamine, 
those in which lactosamine is present at the non-reducing end of the 
oligosaccharide are preferred. In the oligosaccharide containing at least 
one lactosamine, there is no special limitation concerning the linkage 
form between sugar residues other than lactosamine. 
The amino sugar residue in lactosamine and the lactosamine oligosaccharide 
is glucosamine residue or N-acetylglucosamine residue. N-Acetylglucosamine 
residue is preferred. 
As for the number of sugar residues, the lactosamine oligosaccharide 
preferably has about 2 to 20 sugar residues, more preferably has 2 to 15 
sugar residues, and especially preferably has 2 to 5 sugar residues. 
When the amino sugar residue in lactosamine is N-acetylglucosamine residue, 
the glycoside linkage from the galactose residue to the 
N-acetylglucosamine residue in lactosamine is preferably .beta.-glycoside 
linkage, more preferably .beta.1.fwdarw.4 glycoside linkage, i.e., 
Gal.beta.1--4GlcNAc. When the basic backbone is the repeating structure of 
lactosamines, the glycoside linkage from the N-acetylglucosamine residue 
to the galactose residue (i.e., the glycoside linkage from lactosamine 
located on the non-reducing end side to lactosamine located on the 
reducing end side in the two adjacent lactosamines) is preferably 
.beta.-glycoside linkage, more preferably .beta.1.fwdarw.3 glycoside 
linkage, i.e., GlcNAc.beta.1--3Gal. 
Lactosamine may have a sialic acid residue and/or a fucose residue. Namely, 
lactosamine may be sialylated and/or fucosylated. In the case of 
sialylated lactosamine, preferably the sialic acid residue is bound to the 
galactose residue through .alpha.-glycoside linkage, and more preferably 
the sialic acid residue is bound to the galactose residue through 
.alpha.2.fwdarw.3 glycoside linkage, i.e., SA.alpha.2--3Gal. The sialic 
acid includes, for example, N-acetylneuraminic acid and 
N-glycolylneuraminic acid. N-Acetylneuraminic acid is preferred. In the 
case of fucosylated lactosamine, preferably the fucose residue is bound to 
the N-acetylglucosamine residue through .alpha.-glycoside linkage, and 
more preferably the fucose residue is bound to the N-acetylglucosamine 
residue through .alpha.1.fwdarw.3 glycoside linkage, i.e., 
Fuc.alpha.1--3GlcNAc. 
It is allowable and preferable that the N-acetylglucosamine residue in 
lactosamine is sulfated. Oligosaccharides containing lactosamine with 
sulfated galactose residue may be used for the present invention provided 
that it contains at least one lactosamine having galactose residue which 
is not sulfated at hydroxyl group at C-6 position. 
Of the lactosamine oligosaccharides, the lactosamine oligosaccharide which 
has a sialic acid residue at its non-reducing end, is apt to be sulfated 
to some extent as compared with the lactosamine oligosaccharide containing 
no sialic acid residue. Further, the lactosamine oligosaccharide which has 
a sulfated N-acetylglucosamine residue adjacent to the reducing-end side 
of a galactose residue that is not sulfated at hydroxyl group at C-6 
position, is apt to be sulfated as compared with the lactosamine 
oligosaccharide which has a non-sulfated N-acetylglucosamine residue 
adjacent to the reducing-end side of the galactose residue that is not 
sulfated at hydroxyl group at C-6 position. Therefore, it is preferable to 
use those having a sulfated N-acetylglucosamine residue (preferably, 
sulfated at hydroxyl group at C-6 position) adjacent to the reducing-end 
side of the galactose residue that is not sulfated at hydroxyl group at 
C-6 position in the lactosamine oligosaccharide to which sulfate group is 
intended to be transferred (introduced). 
Further, the lactosamine oligosaccharide which is not fucosylated, is apt 
to be sulfated as compared with the lactosamine oligosaccharide having a 
fucosylated N-acetylglucosamine residue. Especially, when the lactosamine 
oligosaccharide has a fucosylated N-acetylglucosamine residue adjacent to 
the reducing-end side of a galactose residue which is not sulfated at 
hydroxyl group at C-6 position (i.e., for example, Gal-(Fuc-)GalNAc), the 
galactose residue is difficult to be sulfated. Therefore, when it is 
intended to obtain a fucosylated sulfated lactosamine oligosaccharide, it 
is preferable that a non-fucosylated lactosamine oligosaccharide is used 
to obtain a sulfated lactosamine oligosaccharide which is then fucosylated 
by using fucosyltransferase. 
It is noted that the lactosamine oligosaccharide to be used in the present 
invention can be appropriately selected by those skilled in the art 
depending on an objective sulfated lactosamine oligosaccharide. 
Specifically, it is preferable to use, as the lactosamine oligosaccharide 
to be used in the present invention, for example, oligosaccharides 
represented by the following formulas: 
EQU Gal--GlcNAc--R (1) 
EQU Gal--GlcNAc(6S)--R (2) 
EQU SA--Gal--GlcNAc--R (3) 
EQU SA--Gal--GlcNAc(6S)--R (4) 
In each of the foregoing formulas, R represents a hydrogen atom or a sugar 
chain containing 1 to 17 sugars, preferably R represents a hydrogen atom 
or a sugar chain containing 1 to 12 sugars, and especially preferably R 
represents a hydrogen atom or a sugar chain containing 1 to 2 sugars. 
When R represents a sugar chain, R preferably includes a basic backbone 
composed of a repeating structure of Gal--GlcNAc. In this case, to the 
basic backbone, for example, any of a sialic acid residue, a fucose 
residue, and a sulfate group may be added. 
It is more preferable to use, as the lactosamine oligosaccharide to be used 
in the present invention, oligosaccharides represented by the following 
formulas: 
EQU Gal--GlcNAc (5) 
EQU Gal--GlcNAc--Gal--GlcNAc (6) 
EQU Gal--GlcNAc(6S)--Gal(6S)--GlcNAc(6S) (7) 
EQU SA--Gal--GlcNAc (8) 
EQU SA--Gal--GlcNAc--Gal--GlcNAc (9) 
EQU SA--Gal--GlcNAc(6S)--Gal(6S)--GlcNAc(6S) (10) 
In each of the foregoing formulas (1) to (10), the sialic acid may be, for 
example, N-acetylneuraminic acid and N-glycolylneuraminic acid. 
N-Acetylneuraminic acid is preferred. 
The source of the lactosamine oligosaccharides usable in the present 
invention is not specifically limited. Any lactosamine oligosaccharide 
obtained by any method can be used, including, for example, those 
extracted and purified from natural products, those produced by chemical 
synthesis, those produced by chemical degradation, those produced by using 
sugar-degrading enzyme, and those produced by using sugar transferase. 
Those commercially available may be used. 
Specifically, in the case of production by using the sugar-degrading 
enzyme, for example, the lactosamine oligosaccharide usable in the present 
invention can be produced by degrading keratan sulfate, preferably keratan 
sulfate obtainable from cartilage, bone, cornea or the like of 
cartilagenous fish such as shark or mammalian such as whale and bovine, 
with, for example, an endo-.beta.-galactosidase type enzyme (for example, 
endo-.beta.-galactosidase and keratanase; any of them is commercially 
available from Seikagaku Corporation) or an endo-.beta.-glucosaminidase 
type enzyme (for example, keratanase II; commercially available from 
Seikagaku Corporation). 
The sulfotransferase which transfers sulfate group to hydroxyl group at C-6 
position of the galactose residue in lactosamine in the lactosamine 
oligosaccharide, is allowed to act on the lactosamine oligosaccharide in 
the co-presence of a sulfate group donor. Thus the sulfate group is 
transferred from the sulfate group donor to hydroxyl group at C-6 position 
of the galactose residue in lactosamine in the lactosamine 
oligosaccharide, and sulfated lactosamine oligosaccharide is produced. 
Preferred sulfated lactosamine oligosaccharides obtainable by the method 
of the present invention include, for example, sulfated lactosamine 
oligosaccharides represented by the following formulas: 
EQU Gal(6S)--GlcNAc-R (11) 
EQU Gal(6S)--GlcNAc(6S)--R (12) 
EQU SA--Gal(6S)--GlcNAc--R (13) 
EQU SA--Gal(6S)--GlcNAc(6S)--R (14) 
In each of the foregoing formulas, R represents a hydrogen atom or a sugar 
chain containing 1 to 17 sugars, preferably R represents a hydrogen atom 
or a sugar chain containing 1 to 12 sugars, and especially preferably R 
represents a hydrogen atom or a sugar chain containing 1 to 2 sugars. 
When R represents a sugar chain, R preferably has a basic backbone composed 
of a repeating structure of Gal--GlcNAc. In this case, to the basic 
backbone, for example, a sialic acid residue, any of a fucose residue, and 
a sulfate group may be added. 
Of the foregoing, it is preferable to use sulfated lactosamine 
oligosaccharides represented by the following formulas: 
EQU Gal(6S)--GlcNAc (15) 
EQU Gal(6S)--GlcNAc--Gal--GlcNAc (16) 
EQU Gal--GlcNAc--Gal(6S)--GlcNAc (17) 
EQU Gal(6S)--GlcNAc--Gal(6S)--GlcNAc (18) 
EQU Gal(6S)--GlcNAc(6S)--Gal(6S)--GlcNAc(6S) (19) 
EQU SA--Gal(6S)--GlcNAc (20) 
EQU SA--Gal(6S)--GlcNAc--Gal--GlcNAc (21) 
EQU SA--Gal--GlcNAc--Gal(6S)--GlcNAc (22) 
EQU SA--Gal(6S)--GlcNAc--Gal(6S)--GlcNAc (23) 
EQU SA--Gal(6S)--GlcNAc(6S)--Gal(6S)--GlcNAc(6S) (24) 
In each of the foregoing formulas (11) to (24), the sialic acid may be, for 
example, N-acetylneuraminic acid and N-glycolylneuraminic acid. 
N-Acetylneuraminic acid is preferred. 
The sulfated lactosamine oligosaccharide (11) is obtained from the 
lactosamine oligosaccharide (1). The sulfated lactosamine oligosaccharide 
(12) is obtained from the lactosamine oligosaccharide (2). The sulfated 
lactosamine oligosaccharide (13) is obtained from the lactosamine 
oligosaccharide (3). The sulfated lactosamine oligosaccharide (14) is 
obtained from the lactosamine oligosaccharide (4). The sulfated 
lactosamine oligosaccharide (15) is obtained from the lactosamine 
oligosaccharide (5). The sulfated lactosamine oligosaccharides (16), (17), 
(18) are obtained from the lactosamine oligosaccharide (6). The sulfated 
lactosamine oligosaccharide (19) is obtained from the lactosamine 
oligosaccharide (7). The sulfated lactosamine oligosaccharide (20) is 
obtained from the lactosamine oligosaccharide (8). The sulfated 
lactosamine oligosaccharides (21), (22), (23) are obtained from the 
lactosamine oligosaccharide (9). The sulfated lactosamine oligosaccharide 
(24) is obtained from the lactosamine oligosaccharide (10). 
The reaction in which the sulfotransferase transferring sulfate group to 
hydroxyl group at C-6 position of the galactose residue in the lactosamine 
oligosaccharide is allowed to act on the lactosamine oligosaccharide, can 
be performed by allowing the sulfotransferase, a sulfate group donor, and 
the lactosamine oligosaccharide to co-exist. In this reaction, pH is not 
specifically limited provided that the activity of the sulfotransferase is 
maintained. However, it is preferable to perform the reaction under a pH 
condition in the vicinity of an optimum reaction pH of the 
sulfotransferase. More preferably, the reaction is performed in a buffer 
having a buffering action at the foregoing pH. The temperature is not 
specifically limited as well provided that the activity of the 
sulfotransferase is maintained. However, it is preferable to perform the 
reaction in the vicinity of an optimum temperature for the 
sulfotransferase. When a substance which serves to increase the activity 
of the sulfotransferase, is available, the substance may be added. The 
reaction time can be appropriately determined by those skilled in the art 
depending on the amounts of the lactosamine oligosaccharide, the sulfate 
group donor, and the sulfotransferase to be used, and other reaction 
conditions. When C6ST is used as the sulfotransferase, for example, the 
reaction is preferably performed in the vicinity of pH 6.4 at a 
temperature in the vicinity of 30 to 40.degree. C., especially in the 
vicinity of 37.degree. C. Further, protamine and/or MnCl.sub.2 may be 
allowed to co-exist during the reaction. 
The sulfate group donor which is used for the reaction to allow the 
sulfotransferase to act, preferably includes activated sulfate 
(3'-phosphoadenosine 5'-phosphosulfate; hereinafter referred to as 
"PAPS"). 
In the case of production in a small amount, it is sufficient that the 
sulfotransferase which transfers the sulfate group to hydroxyl group at 
C-6 position of the galactose residue in the lactosamine oligosaccharide, 
is allowed to exist and make the action of the enzyme in the co-presence 
of the lactosamine oligosaccharide and the sulfate group donor. However, 
in the case of production in a large amount, it is possible to allow the 
enzyme to act continuously by using, for example, immobilized enzyme 
obtained by binding the sulfotransferase to an appropriate solid phase 
(beads or the like), or by using a membrane-type reactor based on the use 
of ultrafiltration membrane, dialysis membrane or the like. Alternatively, 
a bioreactor may be combined and used for reproducing (synthesizing) the 
sulfate group donor. 
In order to recover the sulfated lactosamine oligosaccharide from the 
reaction solution, it is possible to use ordinary methods for separation 
and purification of sugar chains. The sulfated lactosamine oligosaccharide 
can be recovered, for example, by means of operation including, for 
example, adsorption chromatography, anion exchange chromatography, 
hydrophobic chromatography, gel filtration, gel permeation chromatography, 
paper electrophoresis, paper chromatography, fractionation with organic 
solvent (preferably, for example, alcohol and acetone), and a combination 
of the foregoing. However, there is no limitation thereto. For example, 
the sulfated lactosamine oligosaccharide can be recovered by adding NaCl 
to a reaction solution containing the sulfated lactosamine oligosaccharide 
produced in accordance with the method of the present invention so that 
the concentration of NaCl is, for example, about 0.2 M, applying an 
obtained solution to an anion exchange column to remove non-adsorbed 
fractions, and eluting an adsorbed fraction with, for example, NaCl at 
about 2.5 M. In this procedure, the adsorbed fraction (sulfated 
lactosamine oligosaccharide) may be eluted with a concentration gradient 
of NaCl. The condition for the elution and other parameters can be 
appropriately determined by those skilled in the art. 
The obtained sulfated lactosamine oligosaccharide can be utilized as an 
intermediate for producing the basic backbone of the sugar chain of 
GlyCAM-1. For this purpose, when the sulfated lactosamine oligosaccharide 
to be used has a non-sialylated galactose residue (for example, those 
represented by the foregoing formulas (11), (12), (15) to (19)), sialic 
acid can be added to the galactose residue by using sialyltransferase. On 
the other hand, when the sulfated lactosamine oligosaccharide to be used 
has a non-fucosylated N-acetylglucosamine residue, fucose may be added 
thereto by using fucosyltransferase. 
GlyCAM-1 and the backbone of the sugar chain thereof are expected to be 
utilized as anti-inflammatory agents. 
EXAMPLES 
The present invention will be explained more specifically below with 
reference to Examples. However, Examples are only illustrative of the 
present invention, to which the present invention is not limited. 
At first, available sources of reagents used in Examples, exemplary 
preparation of chondroitin 6-sulfotransferase (C6ST), and procedures used 
in Examples will be described below. 
(1) Available Sources of Reagents 
H.sub.2.sup.35 SO.sub.4 : du Pont/NEN; 
.sup.3 H!NaBH.sub.4 (16.3 GBq/mmol) (Amersham); 
PAPS (Sigma); 
Fast Desalting Column HR 10/10 (Pharmacia); 
Hiload Superdex 30 16/60 (Pharmacia); 
Chondroitinase ACII (Seikagaku Corporation); 
Neuraminidase originating from Streptococcus (Seikagaku Corporation); 
.beta.-Galactosidase originating from Streptococcus (Seikagaku 
Corporation); 
Partisil 10-SAX column (Whatman); 
NeuAc.alpha.2--3Gal.beta.1--4(Fuc.alpha.1-3)GlcNAc (SLe.sup.X) (Funakoshi); 
NeuAc.alpha.2--3Gal.beta.1--4GlcNAc (SLN) (Funakoshi); 
Gal.beta.1--4GlcNAc (LN) (Funakoshi); 
Keratan sulfate originating from bovine cornea (Seikagaku Corporation); 
NeuAc.alpha.2--3Gal.beta.1--4GlcNAc(6S) 
.beta.1--3Gal(6S).beta.1--4GlcNAc(6S) (SL2L4) (Seikagaku Corporation); 
NeuAc.alpha.2--3Gal.beta.1--4GlcNAc.beta.1--3Gal.beta.1--4GlcNAc (SL1L1) 
(Seikagaku Corporation); 
Gal.crclbar.1--4GlcNAc.beta.1--3Gal.beta.1--4GlcNAc (L1L1) (Seikagaku 
Corporation); 
.sup.35 S!PAPS (obtained in accordance with Delfert, D. M. and Conrad, H. 
E. (1985), Anal, Biochem., 148, 303-310); 
.sup.3 H!Gal(6S).beta.1--4AManR and .sup.3 H!Gal.beta.1--4AManR(6S) 
(herein referred to as "standard sulfated disaccharides" as well, wherein 
AManR means alditol of 2,5-anhydro-D-mannose) to be used as standard 
substances for high-performance liquid chromatography (HPLC) based on the 
use of Partisil 10-SAX column were obtained by allowing keratan sulfate to 
undergo N-deacetylation, deaminative cleavage, and reduction with 
NaB.sup.3 H.sub.4 to prepare .sup.3 H!Gal(6S).beta.1--4AManR(6S) which 
was then subjected to partial acid hydrolysis (0.1 M HCl, 100.degree. C., 
40 minutes) (see Shaklee, P. N. and Conrad, H. E. (1986), Biochem. J., 
235, 225-236). .sup.3 H!Gal(6S).beta.1--4AManR and .sup.3 
H!Gal.beta.1--4AManR(6S) were purified from products obtained by the 
hydrolysis described above, by means of paper chromatography (developing 
solvent: 1-butanol/acetic acid/1 M NH.sub.3 =3:2:1 (v/v/v) and paper 
electrophoresis; 
Gal.beta.1--4GlcNAc(6S).beta.1--3Gal(6S).beta.1--4GlcNAc(6S) (L2L4) was 
prepared by digesting SL2L4 with neuraminidase. Products obtained by the 
neuraminidase digestion were applied to a column of Partisil 10-SAX, 
followed by elution with a concentration gradient of KH.sub.2 PO.sub.4 
ranging from 25 mM to 500 mM. A peak fraction eluted from the column was 
further purified by using gel filtration chromatography (Superdex 30 
chromatography), followed by lyophilization. 
(2) Preparation of Chondroitin 6-sulfotransferase (C6ST) 
Chondroitin 6-sulfotransferase was purified from a serum-free medium 
obtained after cultivation of chick chondrocyte therein, in accordance 
with a known method (Habuchi, O., Matsui, Y., Kotoya, Y., Aoyama, Y., 
Yasuda, Y., and Noda, M. (1993), J. Biol. Chem., 268, 21968-21974). 
Chondrocytes of chick embryo were inoculated in a culture dish to give a 
concentration of 5.6.times.10.sup.4 /dish, and they were cultured for 11 
days under a condition of 7% CO.sub.2 and 93% air at 38.degree. C. in 
Dulbecco's Modified Eagle's Medium (DMEM) adjusted at pH 7.0, containing 2 
g/L of D-glucose, 100 units/ml of penicillin, 50 .mu.g/ml of streptomycin, 
and 10% fetal bovine serum (FBS). The medium was replaced with a fresh 
medium at pH 7.4 on 2nd, 4th, 7th, 9th, and 10th days after the start of 
the cultivation. 
A medium containing 10% heat-inactivated serum prepared by heating FBS at 
60.degree. C. for 60 minutes was used on 10th day. The cells grew on 11th 
day up to a concentration of 5.0.times.10.sup.6 cells/dish. After that, 
Cosmedium-001 (purchased from CosmoBio) supplemented with 50 .mu.g/ml of 
sodium ascorbate was used to continue cultivation for 10 days while 
exchanging the medium every day. 
The used medium of Cosmedium-001 was collected, and the collected medium 
was centrifuged at 10,000.times. g for 10 minutes. An obtained supernatant 
was adjusted to have a composition comprising 10 mM Tris-HCl, pH 7.2, 0.1% 
Triton X-100, 20 mM MgCl.sub.2, 10 mM 2-mercaptoethanol, and 20% glycerol. 
The culture supernatant was applied to a column of Heparin-Sepharose CL-6B 
(produced by Pharmacia LKB Biotechnology, 2.2.times.28 cm) equilibrated 
with buffer A (10 mM Tris-HCl, pH 7.2, 0.1% Triton X-100, 20 mM 
MgCl.sub.2, 2 mM CaCl.sub.2, 10 mM 2-mercaptoethanol, and 20% glycerol) 
containing 0.15 M NaCl. The column was washed with buffer A containing 
0.15 M NaCl, followed by elution with buffer A containing 0.45 M NaCl to 
perform fractionation. 
Fractions having the sulfotransferase activity were collected, and an 
obtained collected fraction was applied to a column of wheat germ 
agglutinin-agarose (produced by Seikagaku Corporation, 1.2.times.15 cm) 
equilibrated with buffer A containing 0.15 M NaCl. The column was washed 
with buffer A (200 ml) containing 0.15 M NaCl, followed by elution with 
buffer A (200 ml) containing 0.15 M NaCl and 0.3 M N-acetylglucosamine. 
Eluted fractions were collected, and an obtained collected fraction was 
dialyzed against buffer A containing 0.05 M NaCl. 
The obtained fraction was applied to a column of 3', 5'-ADP-agarose 
(produced by Sigma, 1.2.times.11.8 cm, 1.9 .mu.mol 3', 5'-ADP/ml gel) 
equilibrated with buffer A containing 0.05 M NaCl. The column was washed 
with buffer A (150 ml) containing 0.05 M NaCl, followed by elution with a 
linear gradient based on buffer A (300 ml) containing 0.05 M NaCl and 
further containing 0 to 0.2 mM 3', 5'-ADP. Fractions having the 
sulfotransferase activity were collected, and an obtained collected 
fraction was dialyzed against buffer A containing 1 M NaCl, and then 
dialyzed against buffer A containing 0.05 M NaCl. 
In the purification steps, the sulfotransferase activity was measured as 
follows. The reaction solution had the following composition. Namely, the 
reaction solution (50 .mu.l) contained 2.5 .mu.mol of imidazol-HCl, pH 
6.8, 1.25 .mu.g of protamine hydrochloride, 0.1 .mu.mol of dithiothreitol, 
25 nmol (as an amount of glucuronic acid) of chondroitin (produced by 
Seikagaku Corporation), 50 pmol of .sup.35 S!PAPS (adenosine 
3'-phosphate, 5'-phosphosulfate), and the enzyme. 
The activity was measured for various glycosaminoglycans as the substrate, 
by using 25 nmol of glycosaminoglycans (as an amount of galactosamine for 
chondroitin sulfate and dermatan sulfate, or as an amount of glucosamine 
for heparan sulfate and keratan sulfate) in place of chondroitin. 
The reaction solution was incubated at 37.degree. C. for 20 minutes in a 
reaction tube, and then the reaction was terminated by immersing the 
reaction tube in boiling water for 1 minute. After the termination of the 
reaction, 0.1 .mu.mol of chondroitin sulfate A (as an amount of glucuronic 
acid) was added as a carrier, and three volumes of ethanol containing 1.3% 
potassium acetate was added thereto to precipitate .sup.35 S-labeled 
polysaccharide. The mixture was centrifuged at 10,000.times. g for 10 
minutes to obtain a precipitate which was then dissolved in 70 .mu.l of 
water. An aliquot (50 .mu.l) of the obtained solution was injected into a 
desalting column equilibrated with 0.1 M NH.sub.4 HCO.sub.3, and eluted 
fractions containing the .sup.35 S-labeled polysaccharide were collected. 
To an aliquot (200 .mu.l) of an obtained collected fraction, 1 ml of a 
scintillation cocktail (Clearsol, produced by nacalai tesque) was added to 
measure .sup.35 S-radioactivity. Thus incorporation of .sup.35 S into the 
polysaccharide was measured. 
An aliquot (400 .mu.l) was dispensed from the residual solution, to which 
800 .mu.l of ethanol containing 1.3% potassium acetate was added, followed 
by mixing. The mixture was placed on ice for 30 minutes, followed by 
centrifugation at 10,000.times. g for 10 minutes to precipitate the 
.sup.35 S-labeled polysaccharide. The precipitate was dissolved in 25 
.mu.l of a buffer containing 0.1 mg/ml of BSA, 0.05 M Tris-acetate, pH 
7.5, and 10 milliunits of chondroitinase ACII (originating from 
Arthrobacter aurescens, produced by Seikagaku Corporation) to perform a 
reaction at 37.degree. C. for 2 hours. The solution after the reaction was 
spotted onto Whatman No. 1 filter paper together with 
2-acetamido-2-deoxy-3-O-(.beta.-D-gluco-4-enopyranosyluronic 
acid)-6-O-sulfo-D-galactose (.DELTA.Di-6S) and 
2-acetamido-2-deoxy-3-O-(.beta.-D-gluco-4-enopyranosyluronic 
acid)-4-O-sulfo-D-galactose (.DELTA.Di-4S) (each 0.1 .mu.mol, each 
produced by Seikagaku Corporation), followed by development for 20 hours 
with 1-butanol/acetic acid/1 M ammonium hydroxide (2:3:1 (V/V/V)). 
Positions of .DELTA.Di-6S and .DELTA.Di-4S were inspected by using an 
ultraviolet lamp respectively. Portions corresponding to them were excised 
from the filter paper respectively, and were then placed in a scintillator 
prepared by dissolving 5 g of diphenyloxazole and 0.25 g of 
dimethyl-1,4-bis(2-(5-phenyloxazole))benzene in 1 L of toluene to measure 
radioactivity. As for a sample obtained by digestion with chondroitinase 
ACII, the radioactivity remained on the starting point on the filter paper 
was not more than 1% of the spotted radioactivity. According to 
incorporation of .sup.35 S into .DELTA.Di-6S and .DELTA.Di-4S, activities 
of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase were 
calculated respectively. The activity to catalyze transfer of 1 pmol 
sulfate group/minute was defined as 1 unit. As a result, the specific 
activity of chondroitin 6-sulfotransferase was 4.3.times.10.sup.5 
units/mg, and the ratio of activities of chondroitin 
4-sulfotransferase/chondroitin 6-sulfotransferase was 0.02. 
C6ST purified as described above formed a single band on sodium dodecyl 
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under a reduced 
condition, and its molecular weight was determined to be about 75,000. The 
molecular weight was about 160,000 as a result of measurement by using 
Superose 12 HR 10/30 gel filtration chromatography (eluent: 10 mM 
Tris-HCl, pH 7.2, 2 M NaCl, 20 mM MgCl.sub.2, 2 mM CaCl.sub.2, 0.1% Triton 
X-100, and 20% glycerol). Therefore, it was suggested that C6ST formed a 
dimer in the presence of 2 M NaCl. 
The sulfotransferase activity was measured for a variety of substrates. As 
a result, it has been demonstrated that C6ST obtained as described above 
transfers the sulfate group to chondroitin originating from squid skin, 
chondroitin sulfate originating from chick embryo cartilage, chondroitin 
sulfate A originating from whale cartilage, chondroitin sulfate C 
originating from shark cartilage, and keratan sulfate originating from 
bovine cornea, but the sulfate group is only scarcely transferred to 
chondroitin sulfate E originating from squid cartilage, dermatan sulfate 
originating from pig skin, and heparan sulfate originating from bovine 
kidney. It has been confirmed by the present inventors that C6ST transfers 
the sulfate group to hydroxyl group at C-6 position of galactose residue 
in the case of keratan sulfate. 
The activity of C6ST of the present invention was increased by protamine 
and MnCl.sub.2 respectively. 
C6ST had an optimum reaction pH of about 6.4 in the measuring system 
described above. 
(3) Methods Used in Examples 
(3-1) Degradation with Neuraminidase and .beta.-galactosidase 
Digestion with neuraminidase was performed by using a reaction mixture (25 
.mu.l) containing an oligosaccharide to which .sup.35 SO.sub.4 had been 
transferred, 2.5 .mu.mol of potassium acetate buffer (pH 6.5), 0.25 
.mu.mol of CaCl.sub.2, and 10 mU of neuraminidase (see Kiyohara, T. et al. 
(1974), Arch. Biochem. Biophys., 164, 575-582). The reaction mixture was 
incubated at 37.degree. C. for 60 minutes. 
Digestion with .beta.-galactosidase was performed by using a reaction 
mixture (50 .mu.l) containing an oligosaccharide from which sialic acid 
had been removed and to which .sup.35 SO.sub.4 had been transferred, 50 
nmol of L1L1 or L2L4, 2.5 .mu.mol of sodium acetate buffer (pH 5.5), and 
10 mU of .beta.-galactosidase (see Kiyohara, T. et al. (1976), J. 
Biochem., 80, 9-17). The reaction mixture was incubated at 37.degree. C. 
for 60 minutes. 
(3-2) Gel Filtration Chromatography and Paper Electrophoresis 
Gel filtration chromatography was performed by using a column of Hiload 
Superdex 30 16/60. The column was equilibrated with 0.2 M NH.sub.4 HCO3, 
and the flow rate was 1 ml/minute. Fractions of 1 ml or 0.5 ml were 
collected, which were then mixed with 4 ml of Clearsol (produced by 
nacalai tesque) to measure the radioactivity. The oligosaccharide was 
monitored on the basis of absorbance at 210 nm. 
Paper electrophoresis was performed at 30 V/cm for 40 minutes or 80 minutes 
by using Whatman No. 3 filter paper (2.5.times.57 cm) in pyridine/acetic 
acid/water (1:10:400 (v/v/v), pH 4). After the paper electrophoresis, the 
filter paper was dried and cut into small pieces of 1.25 cm and the 
radioactivity was analyzed by means of liquid scintillation counting by 
using a scintillation solution containing 5 g of diphenyloxazole and 0.25 
g of dimethyl-1,4-bis(2-(5-phenyloxazole))benzene in 1 L of toluene. 
(3-3) Quantitative Determination for Glucosamine and Sialic Acid 
The glucosamine content in oligosaccharide was quantitatively determined in 
accordance with a partially modified Elson-Morgan method described by 
Strominger et al. (Strominger, J. L. et al. (1959), J. Biol. Chem., 234, 
3263-3268), after hydrolyzing glycosaminoglycan in 6 M HCl at 100.degree. 
C. for 4 hours. 
Sialic acid was quantitatively determined in accordance with the 
thiobarbituric acid method (Aminoff, D. (1961), Biochem. J., 81, 384-392), 
after performing hydrolysis in 0.1 M H.sub.2 SO.sub.4 at 80.degree. C. for 
60 minutes. 
Example 1 
Transfer Reaction of Sulfate Group to Various Lactosamine Oligosaccharides 
by Using Sulfotransferase 
Structures and abbreviations of lactosamine oligosaccharides used herein 
are shown in Table 1. 
Specifically, the transfer reaction of sulfate group (.sup.35 SO4 was used 
as sulfate group in order to confirm sulfation) to various lactosamine 
oligosaccharides by using sulfotransferase was performed in accordance 
with the following method. A standard reaction mixture was a solution 
containing 2.5 .mu.mol of imidazole-HCl (pH 6.8), 0.25 .mu.mol of 
CaCl.sub.2, 0.1 .mu.mol of dithiothreitol, 0.025 .mu.mol of various 
lactosamine oligosaccharides, 25 pmol of .sup.35 S!PAPS (about 
2.5.times.10.sup.5 cpm), and 0.09 .mu.g of purified chondroitin 
6-sulfotransferase in a final volume of 50 .mu.l. The reaction mixture was 
subjected to the reaction at 37.degree. C. for 60 minutes in a reaction 
tube. The reaction was terminated by immersing the reaction tube in 
boiling water for 1 minute. After the termination of the reaction, 
lactosamine oligosaccharide (sulfated lactosamine oligosaccharide) to 
which .sup.35 SO.sub.4 had been transferred was separated from free 
.sup.35 SO.sub.4 and .sup.35 S!PAPS by means of gel filtration 
chromatography. A fraction (1 ml) was collected to measure the 
radioactivity. 
Results are shown in FIG. 1. A result obtained by using LN as the 
lactosamine oligosaccharide is shown in A, a result obtained by using SLN 
is shown in B, a result obtained by using SLe.sup.X is shown in C, a 
result obtained by using L1L1 is shown in D, a result obtained by using 
SL1L1 is shown in E, a result obtained by using L2L4 is shown in F, and a 
result obtained by using SL2L4 is shown in G. In A to C, open circles 
indicate the results obtained by using purified chondroitin 
6-sulfotransferase having been heat-inactivated by means of a treatment at 
100.degree. C. for 2 minutes (control), and closed circles indicate the 
results obtained by using intact purified chondroitin 6-sulfotransferase. 
In D to G, closed circles indicate values obtained by subtracting values 
obtained by using purified chondroitin 6-sulfotransferase having been 
heat-inactivated by a treatment at 100.degree. C. for 2 minutes (control), 
from values obtained by using intact purified chondroitin 
6-sulfotransferase. 
The retention time of the sulfated lactosamine oligosaccharide was faster 
by 2 to 3 minutes than the retention time (indicated by arrows shown in 
FIG. 1) of the lactosamine oligosaccharide used (lactosamine 
oligosaccharide before sulfation (acceptor)). When SLe.sup.X was used as 
the acceptor, it was predicted that sulfated SLe.sup.X had a retention 
time of 83 to 84 minutes. However, no radioactivity was detected in the 
vicinity of the predicted retention time. Incorporation of sulfate group 
into the lactosamine oligosaccharides is shown in Table 1. A peak of 
sulfated LN partially overlapped with those of free .sup.35 SO.sub.4 and 
.sup.35 S!PAPS. A radioactivity peak (indicated by a horizontal bar in 
FIG. 1A) of a product produced from LN was subjected to paper 
electrophoresis. As a result, the sulfated LN was separated from .sup.35 
SO.sub.4 and .sup.35 S!PAPS. Therefore, incorporation of sulfate group 
(.sup.35 SO.sub.4) into LN was calculated after the paper electrophoresis. 
TABLE 1 
__________________________________________________________________________ 
Abbreviation 
Structure of oligosaccharide Incorporation of .sup.25 S*.sup.1 
__________________________________________________________________________ 
LN Gal.beta.1--4GlcNAc 0.012 
SLN NeuAc.alpha.2--3Gal.beta.1--4GlcNAc 
0.022 
SLe.sup.x 
NeuAc.alpha.2--3Gal.beta.1--4(Fuc.alpha.1-3)GlcNAc 
--*.sup.2 
L1L1 Gal.beta.1--4GlcNAc.beta.1--3Gal.beta.1--4GlcNAc 
0.031 
SL1L1 NeuAc.alpha.2--3Gal.beta.1--4GlcNAc.beta.1--3Gal.beta.1--4GlcNAc 
0.034 
L2L4 Gal.beta.1--4GlcNAc(6S).beta.1--3Gal(6S).beta.1--4GlcNAc(6S) 
0.29 
SL2L4 NeuAc.alpha.2--3Gal.beta.1--4GlcNAc(6S).beta.1--3Gal(6S).beta.1--4Gl 
cNAc(6S) 0.48 
__________________________________________________________________________ 
*.sup.1 pmol/minute/.mu.g protein 
*.sup.2 this symbol indicates no detection. 
According to Table 1, transfer (incorporation) of sulfate group was not 
detected for SLe.sup.X, while incorporation of sulfate group was observed 
for LN, SLN, L1L1, SL1L1, L2L4, and SL2L4. The amounts of incorporation 
into oligosaccharides having sialic acid at their non-reducing ends were 
somewhat larger than the amounts of incorporation into oligosaccharides 
having no sialic acid. Among these oligosaccharides, the maximum 
incorporation of sulfate group was observed in SL2L4. According to this 
fact, it is suggested that the sulfate group (6S) of the GlcNAc(6S) 
residue adjacent to the reducing-end side of the Gal residue increases the 
incorporation of sulfate group into the Gal residue. No incorporation of 
sulfate group was observed for SLe.sup.X. Accordingly, it has been 
suggested that the Fuc residue linked to the GlcNAc residue adjacent to 
the reducing-end side of the Gal residue restrains the transfer reaction 
of sulfate group to the Gal residue by the aid of chondroitin 
6-sulfotransferase. 
Example 2 
Investigation of Position of Sulfation in Lactosamine Oligosaccharide 
In order to determine the position of transfer of sulfate group in the 
sulfated lactosamine oligosaccharide, sulfated SLN was subjected to 
reactions in an order of digestion with neuraminidase, N-deacetylation, 
deamination, and reduction with NaBH.sub.4. An obtained degraded product 
was compared with the standard sulfated disaccharides. Specifically, the 
procedure was as follows. 
SLN including transferred sulfate group (.sup.35 SO.sub.4) (sulfated SLN) 
was prepared in the same manner as the transfer reaction described in 
Example 1 except that the concentration of .sup.35 S!PAPS in the reaction 
mixture was increased 4-fold, and the incubation was performed for 16 
hours. SLN including transferred .sup.35 SO.sub.4 was applied to a column 
of gel filtration (Superdex 30 column). Sulfated SLN eluted from the 
column was lyophilized, purified by means of paper electrophoresis, and 
digested with neuraminidase in the same manner as described above. 
The sample obtained after the neuraminidase digestion was subjected to 
separation by means of paper electrophoresis, followed by hydrazinolysis. 
The hydrazinolysis was performed in accordance with a method described by 
Guo, Y. and Conrad, H. E. (1989), Anal. Biochem., 176, 96-104. The 
desialylated sample was placed into a vial having a volume of 100 .mu.l 
(Reacti-Vial, produced by Pierce), dried in N.sub.2 stream, and finally 
dissolved in 100 .mu.l of 70% hydrazine containing 0.2 mg of hydrazine 
sulfate. The sample was covered with a cap, followed by being left to 
stand in a sand bath at 95.degree. C. for 6 hours. The sample was cooled, 
dried in N.sub.2 stream, dissolved in a small amount of water, and 
lyophilized to remove almost all hydrazine. 
A deacetylated material thus obtained was purified by means of gel 
filtration chromatography and paper electrophoresis, followed by 
deamination with nitrous acid. Specifically, the deamination was performed 
as follows in accordance with a method described by Shaklee, P. N. and 
Conrad, H. E. (1986), Biochem. J., 235, 225-236. Namely, the deacetylated 
material was dissolved in 20 .mu.l of HNO.sub.2 solution (pH 4) (prepared 
by mixing 250 .mu.l of 5.5 M NaNO.sub.2 and 100 .mu.l of 1 M H.sub.2 
SO.sub.4). The sample was left to stand at room temperature for 30 
minutes, and then cooled in ice. pH was adjusted to be 8.5 with 7 .mu.l of 
1 M Na.sub.2 CO.sub.3. The sample was mixed with 10 .mu.l of 0.5 M 
NaBH.sub.4 dissolved in 0.2 M Na.sub.2 CO.sub.3 (pH 10.2) (reduction with 
NaBH.sub.4). Excessive NaBH.sub.4 was degraded by adding 5 .mu.l of 3 M 
acetic acid. The sample was dried in N.sub.2 stream, dissolved in water 
again, and dried again. Finally, the sample was dissolved in 60 .mu.l of 
water, and purified by means of gel filtration chromatography and paper 
electrophoresis. 
Reaction products obtained in the foregoing respective steps were subjected 
to paper electrophoresis to measure radioactivity. Results are shown in 
FIG. 2. In FIG. 2, A shows a result for SLN including transferred .sup.35 
SO.sub.4, B shows a result for a reaction product obtained by digestion of 
a peak fraction shown in A with neuraminidase, C shows a result for a 
reaction product obtained by hydrazinolysis (N-deacetylation) of a peak 
fraction shown in B, and D shows a result for a reaction product obtained 
by deamination of a fraction corresponding to a slowly moving peak 
(indicated by a horizontal bar in FIG. 2C) shown in C, followed by 
reduction with NaBH.sub.4. The results were obtained by performing 
electrophoresis for 40 minutes (A and B) or for 80 minutes (C and D). 
It is assumed that a fast moving peak in FIG. 2C (peak not indicated by the 
horizontal bar in FIG. 2C) corresponds to an unreacted substance brought 
about by incomplete N-deacetylation. 
A peak of a substance obtained after the deamination and the reduction with 
NaBH.sub.4 (indicated by a horizontal bar shown in FIG. 2D) was mixed with 
the standard sulfated disaccharides to perform high-performance liquid 
chromatography (HPLC) by using a column of Whatman Partisil 10-SAX 
(4.5.times.25 cm) equilibrated with 5 mM KH.sub.2 PO.sub.4. The column was 
developed with 5 mM KH.sub.2 PO.sub.4. The flow rate was 1 ml/minute, and 
the temperature of the column was 40.degree. C. Fractionation was 
performed with each fraction volume of 0.5 ml. Obtained fractions were 
mixed with 4 ml of Clearsol respectively to quantitatively measure the 
radioactivity. Results are shown in FIG. 3. In FIG. 3, open circles 
indicate the radioactivity of .sup.3 H, and closed circles indicate the 
radioactivity of .sup.35 S. Peaks 1 and 2 are .sup.3 
H!Gal(6S).beta.1--4AManR and .sup.3 H!Gal.beta.1--4AManR(6S) (standard 
sulfated disaccharides) respectively (Shaklee, P. N. and Conrad, H. E. 
(1986), Biochem. J., 235, 225-236). 
According to FIG. 3, it was demonstrated that major .sup.35 S-radioactivity 
was eluted together with .sup.3 H!Gal(6S).beta.1.times.4AManR, however, 
no .sup.35 S-radioactivity peak was detected at the position of .sup.3 
H!Gal.beta.1--4AManR(6S). Therefore, it has been demonstrated that the 
sulfate group is transferred to hydroxyl group at C-6 position of the Gal 
residue by chondroitin 6-sulfotransferase, however, the sulfate group is 
not transferred to hydroxyl group at 0-6 position of the GlcNAc residue. 
Example 3 
Sensitivity of SL1L1 Including Transferred .sup.35 SO.sub.4 and SL2L4 
Including Transferred .sup.35 SO.sub.4 to Digestion with 
.beta.-Galactosidase 
In order to obtain information on the position of the sulfate group 
transferred to SL1L1 and SL2L4, the sensitivity to digestion with 
.beta.-galactosidase was investigated for SL1L1 including transferred 
.sup.35 SO.sub.4 and SL2L4 including transferred .sup.35 SO.sub.4. 
SL1L1 including transferred .sup.35 SO.sub.4 or SL2L4 including transferred 
.sup.35 SO.sub.4 was digested with neuraminidase in order to remove 
terminal sialic acid. Desialylated substances were separated by means of 
paper electrophoresis. After the electrophoresis, the desialylated 
products originating from SL1L1 including transferred .sup.35 SO.sub.4 and 
SL2L4 including transferred .sup.35 SO.sub.4 were eluted from the filter 
paper, lyophilized, mixed with non-radioactive L1L1 and non-radioactive 
L2L4, respectively, and digested with .beta.-galactosidase. 
The mixture of the desialylated product including transferred .sup.35 
SO.sub.4 and the non-radioactive oligosaccharide was subjected to gel 
filtration chromatography before or after the digestion with 
.beta.-galactosidase. Eluted fractions from the gel filtration column were 
monitored on the basis of the absorbance at 210 nm and the radioactivity 
of .sup.35 S. FIG. 4 shows results for the mixture of the desialylated 
product of SL1L1 including transferred .sup.35 SO.sub.4 and the 
non-radioactive L1L1 . FIG. 5 shows results for the mixture of the 
desialylated product of SL2L4 including transferred 35SO.sub.4 and the 
non-radioactive L2L4. In FIGS. 4 and 5, A and C show the results obtained 
before the digestion with .beta.-galactosidase, and B and D show the 
results obtained after the digestion with .beta.-galactosidase. In FIGS. 4 
and 5, C and D show the absorbance at 210 nm, and A and B show the 
radioactivity of 0.5 ml fractions. 
With reference to the results obtained before the digestion with 
.beta.-galactosidase, the desialylated products originating from SL1L1 and 
SL2L4 including transferred .sup.35 SO.sub.4 were eluted at positions of 
sulfated L1L1 (FIG. 4A) and sulfated L2L4 (FIG. 5A) respectively. This 
fact demonstrated that the desialylation proceeded completely. 
After the digestion with .beta.-galactosidase of the mixture of the 
desialylated product of SL1L1 including transferred .sup.35 SO.sub.4 and 
the non-radioactive L1L1 , the absorbance at 210 nm originating from the 
non-radioactive L1L1 was completely migrated to a slower eluting position 
(FIG. 4D). On the other hand, about 1/3 of the .sup.35 S-radioactivity was 
still eluted at the position of sulfated L1L1 (FIG. 4B). These results 
suggested that about 1/3 of .sup.35 SO.sub.4 transferred to SL1L1 was 
positioned at the Gal residue located on the non-reducing-end side. 
Sulfated L1L1 including transferred .sup.35 SO.sub.4 was prepared and 
digested with .beta.-galactosidase. As a result, no significant change 
occurred in the ratio of the substances including transferred .sup.35 
SO.sub.4 resistant to .beta.-galactosidase (data are not shown herein). 
These results suggest that sialic acid located on the non-reducing end 
does not affect the distribution of transferred sulfate group. On the 
contrary, the desialylated product originating from SL2L4 including 
transferred .sup.35 SO.sub.4 was generally insensitive to 
.beta.-galactosidase (FIG. 5B), while non-radioactive L2L4 was completely 
degraded (FIG. 5D). According to these results, it has been demonstrated 
that all sulfate groups transferred to SL2L4 are located on the Gal 
residue located on the non-reducing end.