Full text of DOI:10.1038/sj.jim.2900818

Journal of Industrial Microbiology & Biotechnology (2000) 24, 262–266
2000 Society for Industrial Microbiology 1367-5435/00 $15.00

www.nature.com/jim
Agglutination of human and animal erythrocytes in marine
unicellular algae
W-R Liao and R Huang
Institute of Oceanography, National Taiwan University, Taipei 100, Taiwan
Ethanol extracts of 12 marine unicellular algae were assayed for agglutinating activity against native and enzyme-
treated human and animal erythrocytes. All of the algae assayed agglutinated at least one group of normal erythro-
cytes from humans. Notably, all algal extracts agglutinated erythrocytes of hemophilia patients arising from coagu-
lation disorders. Meanwhile, all algae had a strong reaction with monkey erythrocytes, but to a lesser extent or not
at all with sheep erythrocytes. Both trypsin and pronase improved the detection of most algal agglutinins and caused
a drastic increase in hemagglutinating activity after treatment for 2 h or more. However, hemagglutinating activity
decreased or disappeared completely when two extracts of different algal species were combined. Journal of Industrial
Microbiology & Biotechnology (2000) 24, 262–266.
Keywords: agglutinating activity; marine unicellular algae; erythrocytes; enzymes
Introduction
Materials and methods
Marine algae contain lectins or agglutinins, which are pro-
teinaceous compounds and have specific binding character-
istics with carbohydrates to produce unique biological
activities [3,8]. Lectins can cause coagulation in human and
animal erythrocytes, bacteria, yeast, blue-green algae and
even mouse tumor cells [4,9,11,16]. They also stimulate
mitogensis of human lymphocytes [12,15]. Although many
marine algae have been assayed for agglutination of human
and animal erythrocytes, those investigations were limited
to macroalgae, ie seaweeds, in a few ecological areas
Algal cultures
Twelve species of marine unicellular algae belonging to
Cyanophyceae, Chlorophyceae, Bacillariophyceae, Dino-
phyceae, Prymnesiophyceae, Eustigmatophyceae and Rho-
dophyceae were used for the hemagglutination assays.
Algal cells were grown xenically in unialgal cultures in an
f/2 enriched seawater medium [10] at 25°C and 15–17 W
−2
m
irradiance of L:D = 12:12 before assaying. The algal
cells used in the assay were in exponential phase (3–5 days
after inoculation in the enriched medium).
[1,2,9,11,17]. Agglutinating activity varies greatly between
algal species and the sources of erythrocytes [1,2,9]. Such
activity can be induced markedly by modifying the extrac-
tion process, ie using enzyme treatments and additives
Preparation of algal materials
Cultures (2–3-litre) of algae were filtered through Whatman
glass filters (GF/C) at low-pressure vacuum, ie 1/2 atm, 2–
[
2,12–14,16]. To date, research on agglutination with
3
drops of Milli Q water (previously distilled) were added
human hemophiliac blood or other coagulation disorders by
marine algae has not been reported. A few species of sea-
weeds have been purified with the biochemical character-
istics of the lectins specified [5,11,17,18]. Rogers and Fish
to the filter to wash out any salt remaining on the cells and
filter. Cells on the filter membrane were then freeze-dried
at −20°C for 48 h. Dried cells on the filter were scraped
down as pellets and weighed. Algal pellets of approxi-
mately 0.2 g immersed in liquid nitrogen were ground into
powder and then kept at −20°C until used.
[17] reviewed the biochemical characteristics of lectins in
various seaweeds and their hemagglutination with human
erythrocytes. Yet lectins and their biochemical character-
istics in unicellular algae remain unknown. Recently, we
found agglutination of human and animal erythrocytes by
the extracts of chrysophycean algae (Liao et al, unpublished
data), revealing that lectins also exist in single-cell algae.
Therefore, we examined more unicellular algae for hemag-
glutination for a better understanding of the biochemical
characteristics of lectins. Additionally, we combined the
extracts from different species and assayed their hemagglut-
ination after combination.
Algal extraction
The extraction procedure followed that of Kawakubo et al
[14]. Ethanol (20%) was added to the algal powder (1:10,
w:v), and the mixture was stirred overnight at 4°C. After
centrifugation, the supernatant mixture was collected for
use in the agglutination assay.
Preparation of erythrocytes
The agglutinating activity of algal extracts was assayed
using fresh human and animal erythrocytes. Some of the
human erythrocytes were normal A, B, O and AB groups,
and others were coagulation disorder (hemophilia) assigned
to failure in activating thrombin within the prothrombin
time (PT) or the activated partial thromboplastin time
(APTT). The disorders arise from the lack of factors VII,
Correspondence: R Huang, Institute of Oceanography, National Taiwan
University, Taipei 100, Taiwan. E-mail: rangȰccms.ntu.edu.tw
Received 24 September 1999; accepted 6 January 2000

Hemagglutination in marine unicellular algae
W-R Liao and R Huang
2
63
Table 1 Hemagglutinating activities of marine unicellular algae against
native and trypsin-treated human erythrocytes; the activity is expressed
as titer
VIII and IX in the blood of patients. Human blood was
obtained from the Union Clinical Laboratory, Taipei, while
monkey and sheep erythrocytes were purchased from
Accurate Chemical & Scientific Corporation, New York.
All of the erythrocytes were concentrated by centrifugation
in a 50 mM phosphate buffer (pH 7.2) containing 0.1 M
sodium chloride [13]. This phosphate-buffered saline (PBS)
was used to dilute the concentrated erythrocytes to 2% of
their original volume. The erythrocytes were then used
directly (native) in the assay for agglutination or treated
with 2% trypsin or 0.5% pronase in PBS at 37°C for 2 h
Algal species
Native
Trypsin
A
B
O
AB
A
B
O
AB
Cynophyceae
Synechococcus sp
2
24
2
1
3
24
7
0
2
2
2
2
2
2
2
Chlorophyceae
Ankistrodesmus sp
Chlorella ellipoidea
2
3
5
1
2
2
1
12
5
1
6
0
0
0
0
2
2
0
2
2
2
2
2
2
2
2
2
5
(enzyme-treated) before using them in assays.
Dinophyceae
Gyrodinium instriatum
Prorocentrum minimum
4
3
1
1
4
4
2
3
4
3
4
3
4
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Hemagglutinating activity assay
Aliquots of algal extracts from serial two-fold dilutions
were added separately to 96-well micro-V-plates. An equal
volume of erythrocyte suspension was added to each well,
mixed with extracts at room temperature and allowed to
stand for 2 h. Hemagglutination was examined and its
activity, which was a reciprocal of the highest two-fold
dilution exhibiting positive agglutination, was recorded.
The control was the aqueous ethanol without algal extracts.
For each species, the assay was carried out in duplicate
Prymnesiophyceae
Hymenomonas sp
Pavlova salina
3
3
5
9
13
12
6
5
6
14
12
7
2
2
2
2
2
2
2
2
0
2
2
2
2
2
2
2
12
12
10
Rhodophyceae
Porphyridium sp
5
4
5
12
7
7
8
2
2
0
2
2
2
2
2
1
Agglutination of hemophilia blood
samples. Activity was determined with an error of ±2
titers.
With the blood of hemophilia patients, all of the algae
assayed agglutinated APTT type erythrocytes, while six of
them reacted positively with PT type erythrocytes (Table
Agglutinating activity of the combined extracts
2
0
2). P. salina demonstrated the strongest activity (2 titers)
Equal volumes of algal extracts from two algal species were
mixed together and shaken. The mixture was placed in
wells for the assay of hemagglutination as described above.
against PT erythrocytes, while Synechococcus sp was
highly active towards both PT and APTT type erythrocytes.
Agglutination of animal erythrocytes
Inhibition test with sugars
Differential preferences for animal erythrocytes were also
distinct in unicellular algae. Nearly all of the algae assayed
reacted strongly with native erythrocytes from monkey, but
not from sheep erythrocytes (Table 3). Monkey erythro-
cytes are more susceptible to algal lectins than sheep
erythrocytes in agglutination.
Sugar (50 ␮l of 0.5 M) in PBS was mixed thoroughly with
an equal volume of algal extract and kept at room tempera-
ture for 30 min. The extracts were assayed again against
native erythrocytes of the human O group to make sure of
their agglutinating activity prior to mixing. Then 50 ␮l of
the mixture was added to a well of a micro-V-plate, mixed
with an equal volume of 2% type O erythrocytes. Aggluti-
nation of erythrocytes was examined after 2 h. Sugars used
in the test were the monosaccharides, d-glucose, d(+)-gala-
cotse, d(+)-mannose, methy-␣-d-glucoside, methyl-␣-d-
mannoside and the oligosaccharides, sucrose, lactose and
raffinose.
Table 2 Hemagglutinating activities of marine unicellular algae against
native and trypsin-treated human coagulation disorder erythrocytes; the
activity is expressed as titer
Algal species
Native
APTT
Trypsin
APTT
PT
PT
Results
Agglutination of human erythrocytes
Cynophyceae
Synechococcus sp
1
2
12
12
14
2
2
2
2
Agglutination of human erythrocytes by marine algae var-
ies greatly among algal species and erythrocytes assayed.
All of the algae assayed agglutinated at least one group of
Chlorophyceae
Ankistrodesmus sp
Chlorella ellipoidea
4
1
5
0
0
2
2
2
2
2
1
2
1
13
4
–25
2
normal erythrocytes, with maximum activity reaching 2
Dinophyceae
Gyrodinium instriatum
Prorocentrum minimum
titers (Table 1). Meanwhile, almost all species agglutinated
strongly native O and AB erythrocytes, but to a lesser
extent for A erythrocytes. The blue-green alga Synecho-
coccus sp and the green alga Ankistrodesmus sp displayed
1
1
1
1
3
3
1
2
2
2
2
2
2
2
1
2
Prymnesiophyceae
Hymenomonas sp
Pavlova salina
12
20
5
1
1
5
4
2
2
2
2
2
2
2
2
4
25
14
extremely strong affinity for O erythrocytes, ie 2 –2
titers, followed by Hymenomonas sp and Pavlova salina,
2
Rhodophyceae
Porphyridium sp
12–13
1
1
2
3
reaching 2
titers. Four species were non-specific for
2
2
2
2
human blood cells.
Journal of Industrial Microbiology & Biotechnology

Hemagglutination in marine unicellular algae
W-R Liao and R Huang
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64
Table 3 Hemagglutinating activities of marine unicellular algae against
animal erythrocytes; N: native, T: trypsin-treated, P: pronase-treated and
the activity is expressed as titer
Hemagglutination of the combined algal extracts
Agglutinating activities of the combined extracts varied
with the species used. However, compared with individual
species, the combined agglutinating activities were strongly
inhibited in general (Table 4). The extremely active extracts
of I. galbana and P. salina (2 titer) against native PT
erythrocytes (Liao et al, unpublished) became inactive after
combination. Similar results of abolished activity occurred
in most of the combinations. In contrast, the combination
of S. costatum with I. galbana resulted in a drastic increase
of agglutination against sheep erythrocytes. Meanwhile, all
of the combined extracts retained high activity against
APTT erythrocytes.
Algal species
Sheep
T
Monkey
T
2
0
N
0
P
N
P
Cynophyceae
Synechococcus sp
6
6
10
18
17
2
2
2
2
2
Chlorophyceae
Ankistrodesmus sp
Chlorella ellipoidea
1
6
1
10
7
11
11
17
18
18
0
0
2
2
2
2
2
2
2
2
2
19
2
Dinophyceae
Gyrodinium instriatum
Prorocentrum minimum
3
3
1
5
8
0
0
0
0
2
2
0
0
2
2
2
0
Inhibition of agglutination
Prymnesiophyceae
Hymenomonas sp
Pavlova salina
1
2
1
4
8
13
17
14
Both mono- and oligosaccharides inhibited algal extracts in
agglutinating erythrocytes (Table 5). However, the inhi-
bition patterns of sugars varied with the algae tested. It
0
0
0
0
2
2
2
2
2
2
2
17
2
Rhodophyceae
Porphyridium sp
2
3
10
7
13
14
2
2
2
2
2
2
Table 4 Agglutination of the combined extracts of two marine algae
against native erythrocytes; the activity is expressed as titer. The numbers
in parentheses indicate the individual activities of two corresponding spec-
ies from Liao et al (unpublished data)
Agglutination with enzyme-treated erythrocytes
Pairs of algal species
Erythrocyte
Pretreatment of erythrocytes with trypsin or pronase caused
an increase of hemagglutinating activity in most of the
assays, particularly in P. salina and Porphyridium sp for A
and PT erythrocytes (Table 1, Figure 1). Meanwhile more
species gave positive hemagglutination reactions after treat-
ment with pronase than with trypsin. Each enzyme also
inhibited markedly algal hemagglutination, eg Ankistro-
desmus sp for O erythrocytes and Hymenomonas sp and P.
salina for PT erythrocytes. The enzyme-induced activity
peaks after treatment for 2 h or more (Figure 1). With ani-
mal erythrocytes, trypsin and pronase also improved detec-
tion, but the latter was superior to the former (Table 3).
B
PT
APTT
Sheep
6
14 20
12
7
Skeletonema costatum
Isochrysis galbana
(0,2 )
(2 ,2 ) (2 , 2 ) (0,0)
12 6 8
8
2
2
2
2
3
14
12 12
S. costatum
Cyclotella sp
(0,2 )
(2 ,0)
0
(2 ,2 ) (0,0)
5
0
2
0
4
14
1
12
1
2
S. costatum
Porphyridium sp
(0,2 )
(2 ,2 )
0
(2 ,2 )
2
(0,2 )
5
2
0
2
6
9
20 20
7
1
I. galbana
Pavlova salina
(2 ,2 )
0
(2 ,2 ) (2 ,2 )
(0,0)
0
5
0
2
Figure 1 Hemagglutination by algal extracts of pronase-treated erythrocytes at various time intervals of incubation (left: Hymenomonas sp with O
erythrocytes, right: Porphyridium sp with PT erythrocytes).
Journal of Industrial Microbiology & Biotechnology

Hemagglutination in marine unicellular algae
W-R Liao and R Huang
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Table 5 Inhibition of algal agglutinating activity against native erythro-
cytes of human O group by sugars
ination inhibition also occurred after enzyme treatment,
particularly with human erythrocytes. Such enzyme inhi-
bition has rarely been mentioned in the literature.
Lectins of seaweeds are generally characterized by
glycoproteins, with or without binding to monosaccharides,
and a requirement for divalent cations [17,18]. They
specifically recognize and bind the carbohydrates in glyco-
proteins. Thus the sugar moiety of glycoproteins is the key
in inhibiting algal hemagglutination. Many glycoproteins,
glycopeptides and monosaccharides or simple sugars, eg d-
Algal species
Monosaccharides
Oligosaccharides
1
2
3
4
5
6
7
8
9
Synechococcus sp
Ankistrodesmus sp
Gyrodinium instriatum
Prorocentrum minimum
Hymenomonas sp
Pavlova salina
−
+
+
+
−
−
+
+
+
−
+
−
+
+
+
+
+
+
+
+
−
−
−
+
−
+
−
−
+
+
+
+
+
+
+
+
−
+
+
−
−
+
+
+
+
+
+
−
+
+
+
+
+
+
−
−
+
+
+
+
+
+
−
+
+
−
+
−
−
−
−
−
glucose,
d-mannose,
d-galactose,
d-fucose,
N-
acetylgalactosamine and N-acetylglucosamine, inhibit
specifically lectins of various seaweeds in hemagglutination
Skeletonema costatum
Cyclotella sp
[
5,11,17,18]. Our study also demonstrated inhibition by
1
: d-glucose; 2: d(+)-galactose; 3: d(+)-mannose; 4: methyl-␣-d-gluco-
monosaccharides and oligosaccharides of the agglutinating
activity of marine microalgae. In view of the photosynthetic
storage food in algal cells, members of Chrysophyceae, eg
the diatoms S. costatum and Cyclotella sp and the flagel-
lates I. galbana and P. salina, have abundant chrysolamina-
ran, a polysaccharide predominated by glucose, and con-
siderable amounts of galactose, mannose, fructose and
fucose [6,7]. Thus the strong hemagglutination inhibition in
the combined extracts of this study probably arose largely if
not entirely from the binding (primary and/or secondary)
of one or more of the above or related simple sugars to the
lectins within two algal species. We do not clearly know
their interactions and binding processes. It appears that dif-
ferent algal combinations exhibited somewhat different pat-
terns of agglutination against the erythrocytes assayed,
implying a variety of cellular carbohydrates (both internal
and external) between species and among taxonomic
groups. Blunden et al [2] and Fabregas et al [9] revealed
that hemagglutinins and their activities in marine algae are
taxonomically significant. Others reported agglutination of
blue-green algae by seaweed extract [11]. There remains
much work to explore the biochemical characteristics of
lectins in unicellular algae, particularly inter- or intraspec-
ies reactions of algal cells with extracellular substances,
with reproduction as well as symbiosis of bacteria or
viruses on algal cells [3,8].
side; 5: methyl-␣-d-mannoside; 6: sucrose; 7: lactose; 8: l-lactose; 9: raf-
finose; + = inhibition, − = noninhibition.
appeared that d(+)-galactose and lactose inhibited all of the
algae tested except Hymenomonas sp and Synechococcus
sp, respectively. G. instriatum and S. costatum remained
active with d(+)-mannose and raffinose, respectively, but
not with others.
Discussion
Like seaweeds, marine unicellular algae contain lectins that
agglutinate human and animal erythrocytes to varying
degrees. In light of their generally strong activities, marine
unicellular algae are also a potent source of lectins for stud-
ies in biochemistry and medical applications.
Lectins of marine macroalgae have generally weak
activity towards human erythrocytes, and are more specific
for A and B than for O erythrocytes [2,4,5,9,16]. In this
study, lectins in unicellular algae displayed comparatively
high activities against human erythrocytes, particularly O
and AB groups. Some of them have particularly interesting
characteristics, eg extremely strong affinity for O erythro-
cytes by lectins from Synechococcus sp and Ankistro-
desmus sp.
The generally strong activity against hemophilia erythro-
cytes in unicellular algae is most interesting since previous
literature has rarely reported similar agglutination by mar-
ine macroalgae. These, together with our preliminary find-
ings with chrysophyte cells (Liao et al, unpublished data)
reveal that lectins which coagulate the anomalous blood,
are widely distributed in marine unicellular algae.
Many authors used erythrocytes from sheep and other
animals to assay hemagglutinins in seaweeds; they found
a range of affinities with the algal species used
Acknowledgements
The authors gratefully acknowledge Shien-Chi Huang of
the Union Clinical Laboratory (UCL), Taipei for providing
human blood samples and Heui-Mei Su of the Tungkang
Fisheries Institute, Taiwan for providing algal cultures.
References
[
1,5,9,12,14]. In our study, most algae reacted strongly
1
Bird KT, TC Chiles, RE Longley, AF Kendrick and MD Kidema.
1993. Agglutinins from marine macroalgae of the southeastern United
States. J Appl Phycol 5: 213–218.
Blunden G, DJ Rogers and WF Farnham. 1978. Haemagglutinins in
British marine algae and their possible taxonomic value. In: Modern
Approaches to the Taxonomy of Red and Brown Algae (Irvine DEG
and JH Price, eds), pp 21–45, Academic Press, London.
against monkey erythrocytes, but to a lesser extent or not
at all with sheep erythrocytes.
Pretreatment of erythrocytes with enzymes, such as
papain, neuraminidase, albumin, trypsin and pronase, can
improve markedly the detection of lectins in seaweeds
2
[
2,16,17]. Such enzyme-dependent lectins are common in
3 Bolwell GP, JA Callow, ME Callow and LV Evans. 1977. Cross-ferti-
lisation in fucoid seaweeds. Nature 268: 626–627.
Chiles TC and KT Bird. 1989. A comparative study of animal erythro-
cyte agglutinins from marine algae. Com Biochem Physiol 94B:
the algae assayed, however, the induced activity varies with
the algal species. Meanwhile, treatment with pronase rather
than trypsin could enhance all algal extracts to agglutinate
both sheep and monkey erythrocytes. However, hemagglut-
4
1
07–111.
5 Chiles TC and KT Bird. 1990. Gracilaria tikvahiae agglutinin. Partial
Journal of Industrial Microbiology & Biotechnology

Hemagglutination in marine unicellular algae
W-R Liao and R Huang
2
66
purification and preliminary characterization of its carbohydrate speci-
ficity. Carbohydr Res 207: 319–326.
nin from the red alga Carpopeltis flabellata. Phytochemistry 26:
1335–1338.
6
7
8
9
Craigie JS. 1974. Storage products. In: Algal Physiology and Biochem-
istry (Stewart WDP, ed), pp 206–235, University of California
Press, Berkeley.
Darley WM. 1977. Biochemical composition. In: The Biology of Dia-
toms (Werner D, ed), pp 198–223, Blackwell Scientific Publications,
Oxford.
Etzler ME. 1986. Distribution and function of plant lectins. In: The
Lectins (Liener IE, N Sharon and IJ Goldstein, eds), pp 371–435, Aca-
demic Press, New York.
Fabregas J, J Liovo and A Munoz. 1985. Hemagglutinins in red sea-
weeds. Bot Mar 28: 517–520.
13 Hori K, Y Shimada, C Oiwa, K Miyazawa and K Ito. 1993. Occurrence
of a novel group of hemagglutinins extractable by pronase treatment
in marine algae. J Appl Phycol 5: 219–223.
14 Kawakubo A, H Makino, J-I Ohnishi, H Hirohara and K Hori. 1997.
The marine red alga Eucheuma serra J. Agardh, a high yielding source
of two isolectins. J Appl Phycol 9: 331–338.
15 Lima HC, FH Costa, AH Sampaio, SA Neves, MB Benevides, DIA
Teixeira, DJ Rogers and ALP Freitas. 1998. Induction and inhibition
of human lymphocyte transformation by the lectin from the red marine
alga Amansia multifida. J Appl Phycol 10: 153–162.
16 Rogers DJ, G Blunden, JA Topliss and MD Guiry. 1980. A survey of
some marine organisms for hemagglutinins. Bot Mar 23: 569–577.
17 Rogers DJ and BC Fish. 1991. Marine algal lectins. In: Lectin Reviews
(Kilpatrick DC, Van E Driessche and TC Bog-Hansen, eds), Vol 1,
pp 129–142, Sigma Chemical Company, St Louis.
1
1
1
0 Guillard RRL and JH Ryther. 1962. Studies of marine planktonic dia-
toms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleve)
Gran. Can J Microbiol 8: 229–239.
1 Hori K, K Miyazawa and K Ito. 1986. Preliminary characterization of
agglutinins from seven marine algal species. Bull Jap Soc Sci Fish 52:
18 Rogers DJ and K Hori. 1993. Marine algal lectins: new developments.
Hydrobiologia 260/261: 589–593.
3
23–331.
2 Hori K, H Matsuda, K Miyazawa and K Ito. 1987. Mitogenic aggluti-
Journal of Industrial Microbiology & Biotechnology