Biochemistry (Moscow), Vol. 69, No. 7, 2004, pp. 770
775. Translated from Biokhimiya, Vol. 69, No.

7, 2004, pp. 945
951.

Original Russian Text Copyright 2004 by Baskova, Zavalova, Basanova, Moshkovskii, Zgoda.
Originally published in Biochemistry (Moscow) On
Line Papers in Press, as Manuscript BM03
283, April 18, 2004.

Protein Profiling of the Medicinal Leech Salivary Gland Secretion

by Proteomic Analytical Methods
I. P. Baskova1*, L. L. Zavalova2, A. V. Basanova1, S. A. Moshkovskii3, and V. G. Zgoda3
1

Faculty of Biology, Lomonosov Moscow State University, Moscow 119899, Russia;

fax: (7
095) 939
1745; E
mail: Saliva1@yandex.ru
2
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho
Maklaya 16/10,
Moscow 119997, Russia; fax: (7
095) 330
6538; E
mail: leech@humgen.siobc.ras.ru
3
Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, ul. Pogodinskaya 10,
Moscow 119992, Russia; fax: (7
095) 245
0857; E
mail: vic@ibmh.msk.su
Received December 15, 2003
Revision received February 3, 2004
AbstractProtein diversity of the high molecular weight fraction (molecular mass > 500 daltons) of salivary grand secretion
of the medicinal leech Hirudo medicinalis has been demonstrated using methods of proteomic analysis. One
dimensional
(1D) electrophoresis revealed the presence of more than 60 bands corresponding to molecular masses ranging from 11 to
483 kD. 2D
electrophoresis revealed more than 100 specific protein spots differing in molecular masses and pI values.
SELDI
mass spectrometry analysis using the ProteinChip System based on chromatography surfaces of strong anion or
weak cation exchanger detected 45 individual compounds of molecular masses ranged from 1.964 to 66.5 kD. Comparison of
SELDI
MS data with protein databases revealed eight known proteins from the medicinal leech. Other masses detected by
proteomic analytical methods may be related to both modifications of known proteins and unknown biologically active com

ponents of leech saliva secretion.
Key words: medicinal leech, salivary gland secretion, proteomics, 1D
electrophoresis, 2D
electrophoresis, protein chip,
SELDI
MS

Salivary gland secretion of the medicinal leech

Hirudo medicinalis exhibits several important functions
required for exclusive feeding of these bloodsuckers.
Secretion components are responsible for suppression of
protecting mechanisms of host organism at the wound;
their functioning allows long
term bloodsucking.
Secretion components are also responsible for inhibition
of blood coagulation by blocking host vascular
platelet
and plasma hemostasis at the wound [1
3]. In the gut of
bloodsuckers, the secretion components maintain antico

agulant and fibrinolytic potential of sucked blood; this
regulates blood digestion and provides antimicrobial pro

tection [4]. Combined action of various components of
the salivary secretion determines the positive effect of
hirudotherapy during treatment of various diseases in
man by skin application of leeches. Salivary gland secre

tion injected into the wound acts on various systems of
the human body directly linked to the hemostatic system.
However, only a few protein components have been iden

tified in the leech secretion [5
13]. So identification of
* To whom correspondence should be addressed.

proteins secreted by the salivary glands and encoded by

the leech genome is still an important problem. Such
information may be obtained using proteomics, which
allows identification, characterization, and quantifica

tion of various proteins in cells based on highly sensitive
methods of separation and analysis [14]. However, lack of
information on medicinal leech genome seriously com

plicated proteomic analysis of salivary gland secretion of
Hirudo medicinalis and so it is impossible to carry out
total proteomic analysis of salivary secretion proteins.
Nevertheless, in the present study we have tried to map
proteins of high molecular mass fraction (>500 daltons)
of salivary secretion.

MATERIALS AND METHODS

Preparation of high molecular weight leech salivary
gland secretion. Salivary gland secretion was obtained as
described previously [15]. Medicinal leeches Hirudo
medicinalis grown at the Girud I.N. biofactory
(Balakovo, Saratov Region, Russia) were starved for at

0006
2979/04/6907
0770 2004 MAIK Nauka / Interperiodica

PROTEIN PROFILING OF LEECH SALIVARY GLAND SECRETION

multiple functions of medicinal leech salivary gland
secretion.
In the present study, we were able to identify proteins
that had previously been isolated from leech extracts and
related to salivary gland secretion. These include tryptase
inhibitor, bdellastasin, hirudin, destabilase
lysozyme,
hyaluronidase, calin, and
glutamyl transpeptidase.
Factor Xa inhibitor previously isolated from the leech
secretion [9] represents the only exception. Its molecular
mass [9] was determined for recombinant (but not native)
protein obtained during mutation of the corresponding
gene. This might explain why it has not been detected
among proteins of salivary gland secretion. It should also
be noted that some other proteins previously isolated
from the medicinal leech (e.g., bdellin B [24], hirustasin
[25], carboxypeptidase inhibitor [26], eglin b and eglin c
[27]) were not found in the salivary gland secretion, pos

sibly due to their low concentration.
Thus, use of the method of proteomic analysis
demonstrated diversity of protein components in high
molecular weight fraction of medicinal leech salivary
gland secretion. Identification of their structures and
functions require further investigation.
This study was supported by the Russian Foundation
for Basic Research (grant No. 03
04
48508).

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