134
BIOCHEMICAL SOCIETY TRANSACTIONS
T h e levels of a-2-~-fucosyltransferasein the platelet preparations and plasma from Mth I1 1 were comparable with those
found in the control samples. A small amount of a - 3 - ~ fucosyltransferase activity, detected in plasma under standard assay conditions with N-acetyl-lactosamine as the
acceptor substrate (Table I), was confirped by increasing the
amount of GDP-[14C]fucosein the reaction mixture and the
time of incubation. This activity was, however, only about
8% of the mean a-3-~-fucosyltransferaselevel in normal
plasma samples. In contrast, the levels of activity detected in
Mth I1 1’s granulocyte preparation was comparable with that
found in the control granulocyte samples. T h e presence of
the platelet contamination in the mononuclear cell preparation prevented strict quantitative comparison with control
samples, but strong a-3-fucosyltransferase activity WBS
detected in these cells. T h e presence of the enzyme in
apparently normal amounts in the leucocytes of this individual, despite the very low level in his plasma, is in accord
with the observations of Caillart et al. (1987) that X-antigen
may be expressed on leucocytes in individuals in whom the
a-3-fucosyltransferase is absent, or greatly reduced, in
plasma.
We thank Dr Phyllis Moores, Natal Blood Transfusion Service,
South Africa for the blood samples from Mth I1 1. P.O.S. is supported by the Leukaemia Research Fund, U.K.
Caillart, T., Le Pendu, J., Ventura, M., Mada, M., Coullin, P. & Oriol,
R. ( I 987) Proc. 9th Ini. Symp. Glycoconjugates, Lille (Montreuil,
J., Verbert, A,, Spik, G. & Fournet, B., eds.), Abstract F19.
Gooi, H. C., Feizi, T., Kapadia, A,, Knowles, B. B., Solter, D. &
Evans,M.(1981) Nature(London) 292, 156-158
Greenwell, P., Johnson, P. H., Edwards, J.-M., Reed, R. M., Moores,
P. P., Bird, A,, Graham, H. A. & Watkins, W. M. ( 1 986) Blood
TransJ Irnrnunohaemntol. 29,233-249
Huang, 1. C., Civin, C. I., Magnani, J. L., Shaper, J. H. & Ginsburg, V.
(1983)Bl00d61,1020-1023
Johnson, P. H. & Watkins, W. M. ( I 982) Biochern. SOC. Trans. 10,
445-446
Johnson, P. H. & Watkins, W. M. ( 1987) /’roc. 9th Inr. Symp. Glycoconjugates Lille (Montreuil J., Verbert. A., Spik, G. & Fournet. B..
eds.), Abstract E I07
Skacel, P. 0.& Watkins, W. M. ( 1 987) GlycoconjiigoleJ. 4,262-272
Received 23 June 1988
Tissue differences in the isoelectric focusing profiles of the human blood group A’ and A* gene
specified glycosyltransferases
PAMELA G R E E N W E L L and WINIFRED M. WATKINS
Divisiori of lnimunochemical Genetics, M R C Cliriicml
Research Centre, Harrow, Middlesex H A I 3UJ, U.K .
T h e human blood group A in the ABO system has two major
subgroups A , and A> which are differentiated serologically
by their reactivity with anti-A, and anti-H reagents (Race &
Sanger, 1975). Both A , and A 2 genes encode a-3-N-acetylgalactosaminyltransferases which catalyse the transfer of N-
acetyl-D-galactosamine from U DP- N-acetyl-D-galactosamine
to H-active structures to form A determinants. T h e A , and
A’ transferases are distinguished by their p H optima, cation
requirements, heat stability, kinetic properties and isoelectric
points (see Watkins, 1980) and by their capacity to utilize
certain acceptor substrates (Clausen et al., 1985).
Earlier isoelectric focusing studies showed that the a-3N-acetylgalactosaminyltransferases in A , sera focused with
major peaks in the p H range 8-9.5 and minor peaks in the
Table 1. Distribution of a-3-N-acetyl-n-gcrlacrosaminylrransferase activity in isoelectric
focused fractions of body fluids and tissue extractsfrom blood group A and A? individuals
Preparative flat-bed isoelectric focusing was carried out on an LKB Multiphor apparatus
with Ultrodex (LKB) as the support medium and Ampholines (LKB) in the pH range
3.5- 10 as the carrier ampholytes. The neuraminidase activity of the ovarian cyst fluids
and tissue extracts was measured by the release of sialic acid from a blood group active
glycoprotein (Pusztai & Morgan, 1961 ). The sialic acid liberated in 1 h was estimated by
the method of Warren (1959).Release of sialic acid from serum and cyst fluids (7 ml) was
effected with 1.: cholercr neuraminidase (Koch-Light) by treatment with 0.3 units for 7 h at
37°C. N.T., neuraminidase treated.
~
Transferase activity
(”%
of total) focusing
in the pH range
Enzyme source
Tissue
Blood group
No. tested
Neuraminidase
activity
Sialic acid released
( O h of total)
3.5-7.0
7.0-10.0
30
80
60
70
20
40
-
30
70
2and 18
75
65
5
48
5
25
35
95
52
95
0
0
16
40
Stomach
10
66
90
34
0
0
Kidney
12
8
88
92
50
50
0
0
1989
627th MEETING. NOTTINGHAM
pH range 5-7, whereas the enzymes in A, sera focused with
major peaks in the pH range 5-7 and minor pcaks in the pH
range 7.5-8.5 (Topping & Watkins, 1975; Greenwell &
Watkins, 1979). Although a minor fraction of A transferase
activity in A , serum had a PI similar to the enzyme in A,
serum, the two species separated from A , serum were found
to have identical properties with respect to pH optimum and
cation requirements. Similarly, the two fractions in A, serum
had the same properties as each other and differed from both
species in A , serum.
The observation that, in contrast to serum, both A I and
A? gene-specified transferases in ovarian cyst fluids had isoelectric points in the region of pH 9 (Greenwell & Watkins,
1979) suggested that one species might arise from the other
by a post-translational modification of the enzymc protein.
Examination of larger numbers of ovarian cyst fluids has
now revealed two distinct patterns for the enzymes in
samples from group A, patients. In one group, the focusing
profiles resembled those found in A? serum samples,
whereas in the second group the pattern was similar to that
seen with A , serum samples (Table I ) . In other properties
measured, the various A transferase species were indistinguishable from each other. Further tissue-related differences in isoelectric points were found on cxamination of
a-3-N-acetylgalactosaminyltransferaseactivity in human
stomach mucosa and kidney. The enzymes in extracts of
group A , stomach mucosa show only one peak with a pT of
9.5-10, whereas the A 2 transferases, in the limitcd number
of samples tested, had profiles similar to those givcn by the
enzymes in A, serum. In contrast, extracts from kidneys
derived from either A , or A, donors had focusing profiles
resembling those givcn by the transferases in A , serum
(Table 1). These observations reinforce the view that the
135
measured isoelectric point of the transferase does not result
directly from the gene-encoded structures of the enzymes,
but from some tissue-dependent modification of the enzyme
molecules which changes the pl without altering the A or A'
gene-associated activity. As reported earlier (Greenwell &
Watkins, 1979), treatment of A, serum with Vibrio cholera
neuraminidase results in a shift in the pl of the a-3-N-acetylgalactosaminyltransferase in A, serum to a more basic pH.
Similar treatment of a group A, ovarian cyst fluid [A, (2)]
changed the proportion of the enzyme focusing between pH
7 and 10 from 35% to 95% (Table 1).Hence changes in the
pattern of sialylation might form the basis for the observed
variations in the isoelectric points of the transferases.
Support for this hypothesis comes from examination of
neuraminidase levels in the various tissues and body fluids
since sources of the A' transferase which yielded enzymes
with the more basic isoelectric points were those exhibiting
strong neuraminidase activity (Table 1).
Clausen, H., Levery, S. B., Nudclman, S. K, Tsuchniya, S. & Hakomori, S. ( 1985) /'roc. Nntl. Acrid. Sci. U.S.A. 82, 1 199- I203
Grcenwell, P. & Watkins, W. M. ( 1 979) 9th In[. Congr. Biochern.,
Toronlo Abstract 03-1-S38. p. 156, National Research Council
of Canada
Pusztai, A. & Morgan, W. T. J. ( I96 I ) Biochern. J. 78, 135- 146
Race. R. R. & Sanger, R. ( I 975) Blood Groirps in Man. 6th edn.
Blackwell, Oxford
Topping, M. & Watkins, W. M. ( I 975) Biochern. Biophys. Hes.
Cornmun. 64,89-96
Warren, L. ( 1959) J. Biol. Chem. 234, 197 1- I975
Watkins, W. M. ( 1980)Adv. Hitrncin Genet. 10, I - 136
Received 20 June 1988
Serum a-fetoprotein and thyroxine-binding globulin in hepatocellular carcinoma
MING-QING DU, WINSTON L. HUTCHINSON,
PHILIP J. JOHNSON and ROGER WILLIAMS
Liver Unit, King's College Hospital School of Medicine and
Deniistry, Denmark Hill, London SE5 8UX, U.K.
Hepatocellular carcinoma (HCC) is one of the major forms
of primary liver cancer and its early diagnosis remains an
outstanding clinical problem. To this end, use has been made
of rising serum levels of certain glycoproteins which often
accompany the onset of the disease. Among these; a-fetoprotein ( A F P ) and, to a lesser extent, thyroxine-binding
globulin (TBG)have received much attention (Sell & Becker,
1978; Ruoslahti & Seppala, 1979; Alexopoulos et al., 1988).
In addition to changes in the levels of serum glycoproteins
during HCC development, it appears that certain structural
features may also be altered. A F P from HCC patients displays markedly increased lectin binding when compared with
the protein from patients with benign liver diseases or
tumours of non-hepatic origin (Ruoslahti et al., 1978;
Buamah et a[.: 1984; Buamah et al., 1986). This property
offers the possibility of an additional means of differentiating
between HCC and other liver diseases, such as cirrhosis, in
which glycoproteins including A F P are sometimes elevated.
Here we investigate further the specificity of these alterations
in serum glycoprotein properties by examining the relative
levels of A F P and TBG in HCC, and additional control
groups of subjects displaying increased serum levels of these
proteins. These were fulminant hepatic failure ( F H F ) and
Abbreviations used: HCC, hepatocellular carcinoma; AFP, afetoprotein; TBG, thyroxine-binding globulin; FHF, fulminant
hepatic failure
Vol. 17
cirrhotic patients on one hand and normal pregnant subjects
on the other. The extent to which A F P from each group
bound to immobilized lentil lectin (fucose-recognizing) was
assessed.
Serum TBG levels among HCC patients ( l o ) , fulminant
hepatic failure/cirrhotic control (8) and pregnant control
subjects (8)were measured by radioimmunoassay according
to previously published methods (Alexopoulos et al., 1988).
AFP was measured using a commercially available enzymelinked immunoassay system (Abbott Diagnostics, Abbott,
Herks, U.K.). The binding of A F P to lentil lectin was
assessed by affinity chromatography of serum samples
(120-500 ng of AFP) on a lentil lectin-Sepharose 4B conjugate (Sigma Chemical Co., Poole, Dorset, U.K.) at 4°C.
Chromatography was carried out in 50 mM-Tris/HCI buffer,
pH 7.4, containing 150 mM-NaCI, 1 mM-CaCI, and 1 mMMgCI,. Elution of bound A F P was achieved using 200 mMa-methyl-D-mannoside dissolved in the chromatography
buffer. Eluted AFP was expressed as a percentage of the
total applied to the column.
Table 1. Charucieristics of serum AFI' und TBG in HCC rind
conirols
Samples
HCC
FHF/cirrhotic
Pregnancy
T B G concn.
(Pdml)
A F P range
35+8
19+5
55f10
390-9 X 10'
300- I700
240-500
(ngiml)
AFP/lectin
reactivity
("A,)
48 k 22
9kx
4f3