Phytochemzstry,

Vol. 32, No. 5, pp. 1209-1211,1993

Printed in Great Britain.

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003l- 9422/93$6.00+ 0.00

0 1993Pergamon Press Ltd

AND CHARACTERIZATION
OF A GALACTOSE-SPECIFIC
LECTINFROM
PSILOCYBEBARRERAE

EVELIA HERNANDEZ, REBECA ORTIZ, FRANCISCO L~)PEz, FELIPE MASO,*

TINAGE? and EDGAR ZENTENO~

LUIS F. MONTARO,* ARLEITE MAR-

Departamento de Biologia Experimental, Universidad Aut6noma de Morelos, Cuemavaca, Morelos, Mexico; *Departamento de
Bioquimica, Instituto National Enfermedades Respiratorias, Tlalpan 14080, Mexico; TInstitute de Recherches sur le Cancer, Lille,

France; fDepartamento de Bioquimica, Facultad de Medicina, U.N.A.M., 04510 Mexico

(Received 16 June 1992)
Key Word Index-Psilocybe

barrerae; mushroom;

plant lectins; macromycetes;

galactose-specific

lectin.

Abstract-A

M, 28 000 galactose-specific lectin from the mushroom Psilocybe barrerae has been isolated and purified
by single step affinity chromatography on a horse red blood cell stroma column. This is the first time that a lectin has
been purified from a plant that contains psychotropic substances.

INTRODUCTION

Table 1. Purification of P.

barrerae

&tin from dried fruit body

(1 g)

Plant Wins have been isolated from several mono- and

dicotyledenous plants [l, 21. Fungi, including edible
mushrooms, are an important source of these carbohydrate-specific proteins [3-81 but so far there are no
reports of a lectin isolated from a fungus, or a plant,
containing psychotropic substances. Here we report the
purification of a lectin isolated from Psilocybe barrerae,
an edible mushroom that contains psilocibine, a powerful
hallucinogen [9].

Crude extract
Affinity chromatography

RFSULIS AND DISCUSSION

Aflinity chromatography on a horse red blood cell

stroma column was used to purify a lectin from a
P. barrerae crude extract; the haemagglutinating activity
was recovered from the column with 0.2 M lactose. The
purification process increased 32-fold the lectin specific
activity (Table 1); the lectin content in the fruit body
corresponds to 0.8% of the dry weight. SDS-PAGE
(denaturing and undenaturing conditions) revealed that
the lectin possesses a subunit of M, 28000 but ultracentrifugation assays on sucrose gradients revealed a M,
of 75 000 with a SWzovalue of 5.3, thus indicating that the
lectin has a tendency to form polymeric links. The amino
acid composition of P. burrerae lectin is listed in Table 2.
The lectin lacks cysteine residues, is rich in glutamic,
glycine, aspartic and serine residues, and is poor in
methionine and histidine residues. The sugar content
corresponded to 9.5% by weight and galactose, glucose,
mannose and xylose are the main carbohydrate components (Table 3). Psilocybe barrerae lectin preferentially
agglutinated horse and rabbit erythrocytes, although
erythrocytes from other animal species including human

Specific
agglutination
activity
(titre mg-
protein)

Total
protein
(mg)

Total
agglutination
activity
(titre)

192

128000

666

32 800

21519

1.52

Titre is defined as the reciprocal of the end-point dilution

exhibiting haemagglutination. Type 0 human erythrocytes were
used for testing.

1209

Table 2. Amino acid composition

barrerae
lectin

of P.
-

Amino acid

mol% Amino acid

mol%

ASX

11.2
5.9
10.1
13.3
4.7
12.8
8.3
0
6.3

0.8
4.0
7.0
3.1
3.7
2.1
3.6
3.4

Thr
Ser
Glx
RO

GUY
Ala
CYS
Val

Met
Be
Leu
Tyr
Phe
His
Lys
Arg

The amino acid composition is expressed as mols per Cent assuming a M, of

28 000.

1210

E. HERK&NDEZ
et al.
Table 3. Carbohydrate composition of P.
barrerae lectin

Table 5. Inhibition of haemagglutination

by glycoproteins

Residues
(mol/mol) *
Rhamnose
Fucose
Galactose
Mannose
Glucose
N-Acetyt-o-glucosamine
Xylose

1.6
0.4
1.3
2.4
3.2
0.5
1.3

*Calculatrons were made assuming a IV,

of 28 000 per subunit.

ABO, were also recognized. Pronase- or neuraminidasetreatment of the red blood cell increased five- to six-fold
the haemagglutinating
activity of the lectin (Table 4).
The lectin sugar specificity was determined by haemagglutination
inhibition
assays (Table 5). D-Galactose,
lactose and sialyllactose (a2,3 or a2,6) at concentrations
of 2.5 mM, inhibited four haemagglutinating
units of
P. barrerae lectin. The inhibition by methyl- and phenyltl- or /?-galactosides (10 mM) indicates that P. barrerae
lectin binding is not influenced by the galactose anomeric
carbon. Psilocybe barrerae lectin sugar specificity seems
to be directed to 0-glycosidically-linked
glycans since the
lectin haemagglutinating
activity was effectively inhibited
by asialo glycophorin and asialo fetuin containing 12 and
three Gal (/II, 3) GalNAc (al, 3) ser/thr O-linked oligosaccharides, respectively [lo, 1 I]. Nevertheless, the specific receptor for P. barrerae lectin seems to be more
complex, since glycoproteins containing oligosaccharides
with blood group A and H activity, such as those from pig
stomach mucin [12] and bovine submaxillary
mucin
[13], were good inhibitors of the lectin haemagglutinaTable 4. Haemagglutinating activity* of P. barrerae lectint
Enzyme treatmenti
Erythrocytes

Native

Pronase

Neuraminidase

Human A,
Human A,
Human B
Human 0
Rabbit
Chicken
Rat
Mouse
Sheep
Horse

32
32
32
32
512
2
16
32
32
512

1024
1024
1024
1024
4096
128
256
256
512
4096

2048
2048
2048
4096
9192
64
128
256
1024
9192

*The haemagglutinatmg titre is reported as the mverse

of the last dilution with agglutinating activity.
tThe lectin concentration was 0.04 mg ml-.
fPronase was from S. griseus and neuraminidase from
V. cholerae.

of P. barrerae lectin

Minimum inhibitor
concentration
(PM)+

Glycoprotein*

Source

Asialo glycophorin

human 0
erythrocytes
pig

0.01

pig
bovine
bovine

0.001
0.1
0.035

Stomach mucin
Stomach mucin glycosylpeptides
Submaxillar mucin
Submaxillar asialomucin
Submaxillar asialoagalact0 mucin
Fetum
Asialofetuin
Ovalbumm
Orosomucoid
Serum transferrin
Milk transferrin
Serum transferrin

bovine
foetal calf
foetal calf
chicken
human
human
human
horse

0.01

30.0
0.2
0.05
N.1.S
N.I.
N.I.
NJ.
N.I.

*o-Galactose, lactose and a2,3- or a2,6+ialyllactose inhibited

at concentrations of 25 mM. Glucose, glucosamine, N-acetylglucosamine, galactosamine, N-acetylgalactosamine,
mannose,
mannosamine, fructose, xylose, ribose, rhamnose and maltose
did not inhibit at concentrations up to 100 mM.
tThese are the minimal concentrations required for inhibiting
the haemagglutination activity of P. barrerae lectin (titre 4).
SN.1.. not inhibitory at a 100 PM concentration.

ting activity. Moreover, the relevance of the galactose

residues in terminal positions was made more evident by
the fact that bovine submaxillary
mucin treated with
fi- galactosidase
lost much of its capacity to inhibit the
lectin haemagglutinating
activity.
Psilocybe barrerae lectin is relatively thermoresistant
as no loss in agglutinating activity was observed after a 72
hr incubation at 65, incubation of the lectin at temperatures over 80 for 15 min abolishes their haemagglutinating activity. Treatment with 1 M acetic acid and 0.1 M
EDTA or addition of divalent cations did not modify the
haemagglutinating
activity, indicating that, similar to
other lectins from fungi [3-81, P. barrerae lectin does not
require metals to sustain its agglutinating
activity.
The affinity of P. barrerae lectin for galactose residues
is similar to that of basidiomycetes
lectins [3-81. Psilocybe barrerae corresponds to the Agarical order and can,
therefore, be compared with Ayaricus bisporus lectin [4].
Due to their requirement for 0-glycosidic structures, we
can presume that, similar to lectins from several plant
species [14], the lectins from basidiomycetes
are wellpreserved proteins. To our knowledge this is the first
report of a lectin purified from an organism that contains
hallucinogenic
substances.
EXPERIMENTAL
Isolation of P. barrerae lectin (PBL) . Psilocybe barrerae was collected in Tetela de1 Monte, Morelos, Mexico

Psilocybebarrerae lectin

and identified at the Centro de Investigaciones Biolbgicas, Universidad Aut6noma de1 Estado de Morelos.
Mushroom (10 g) was homogenized with 100 ml isotonic
saline soln (0.9% NaCl;SSI) by continuous stirring at 4
for 24 hr. The supernatant was filtered through a Whatman No. 1 paper filter containing 1 g activated charcoal.
The clear filtrate (crude extract) was poured on to a
column (3 x 30 cm) containing horse red blood cell
stroma physically immobilized on Sephadex G-25, previously equilibrated with SSI at room temp [lS]. The
elution of the lectin was performed with 0.2 M lactose in
SSI at room temp. The eluate was coned in an Amicon
cell with a PM-10 membrane and dialysed against SSI
before storing at 4 until use.
Analytical methods. Protein concn was determined by
the method of Lowry [16] with BSA as standard.
Carbohydrate
content
was determined
by the
PhOH-H,SO,
method [17] with lactose as standard.
The quantitative composition of sugars was determined
by GC as trifluoracetyl alditol derivatives after methanolysis, using mesoinositol as int. standard [18]. Purified lectin (100 pg) was subjected to hydrolysis for 24 hr
under vacuum with 2 ml 6 M HCl at 110 in a sealed
container and its amino acid composition determined in a
Durrum amino acid analyser. The lectin M, was determined by SDS-PAGE using 10% gels according to
Laemmli [19]. The native lectin M, was determined by
ultra-centrifugation
experiments in linear sucrose gradients [20] using a Beckman LS-65 ultracentrifuge. The
sedimentation coefficient was determined according to
Martin and Ames [21].
Haemagglutination assays. The haemagglutinating activity was tested in microtitre U plates (NUNC, Denmark) according to a 2-fold serial dilution procedure with
2% (w/v) untreated erythrocyte suspension in SSI, or
with neuraminidase-treated (v. cholerae fraction V; 0.1 U
0.5 ml- of packed erythrocytes at 37 for 30 min), or
pronase-treated ( S. griseus fraction XXV; 100 pg 0.5 ml-
of packed erythrocytes at 37 for 30 min) red blood cells.
The haemagglutinating titre is reported as the inverse of
the last dilution with agglutinating activity. The sugar
specificity of the lectin was tested by the inhibition of the
hacmagglutinating
activity [22] using simple sugars,
glycosides and glycoproteins. Sialic acid was liberated
from glycoproteins by acid hydrolysis according to ref.
[ 111. Asialo bovine submaxillary mucin was treated with
/$galactosidase from bovine testis (0.1 U PM- of galactose) [23] ; the results are expressed as the minimal concn
of carbohydrates and glycoproteins with well known
structure that effectively inhibited 4 haemagglutinating
dose units of the lectin.
Physicochemical properties. Purified P. barrerae lectin
was exposed to a wide range of temps for various periods
of time, before assessing its haemagglutinating activity. In
order to determine the effect of cations on the lectin
agglutinating activity, we first incubated the lectin with 1
M HOAc followed by treatment with 0.1 M (w/v) EDTA;
after extensive dialysis against deionized H,O lectin

1211

activity was assayed in the presence or absence of 5 mM

CaCl,, MgCl, and MnCl,.
Acknowledgements-The
authors would like to express
their gratitude to the Ministaire Francaise des Affairs
Etrangeres for financially supporting this project.

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