Balasubramanian, A. S.

Job, C. K.

Satish, P. R.

Suneetha, L. M.

Suneetha, S.

English

Publications of the IAS Fellows

INII (RN.\I Ic )\: SI. JI)t'R\AI. c )I' LP.PK1)SY
^
Volume 65, Na)ll1hcr 3
Prime t in lhe
(ISSN (ti-18-916X )
M. leprae Binds to a 28-30-kDa Phosphorylated
Glycoprotein of Rat Peripheral Nerve 1
Lavanya M. Suneetha, Posettihalli R. Satish, Sujai Suneetha,
Charles K. Job, and Aiyalam S. Balasubramanian''
Many pathogenic hacteria specifically ,
recognize anel bind to the carbohydrale
ntoicties of the host ccll surface ('). Que of
the hallntarks of leprosy is the infection of
peripheral nerves hy Mvrohw'ierium lepru u'
(').'"). Recent studies in nasal epithelial cclls
have shown that M. leprae bina to the ex-
tracellular matriz protein fibronectin (FN).
This, in tora, hinds to integrin FN receptors
which help in the internalitation of the
bacilli ( 2 .''). 1-towever, little is understood
about the M. leprae -peripheral nerve pro-
tela interaction.
Protein phosphorylation is a post-transla-
tional ntodification of proteins important in
signal transduction and is especially pronti-
nent in nerve tissues ('•'). Recent studies
have shown the involventent of phosphory-
lation events in the hinding of . bacteria to
the host cells ('•'"•")
[arlicr studies from our lahoratory have
shown that puritied M. /eprue couta inhibit
the phosphorylation of a 28-30-kDa pro-
tein of the rat peripheral nerve and of a 25-
kDa protein of the huntan peripheral nerve
One of the mechanisms proposed for
the observation was ali interaction between
M. leprae and the phosphorylated proteins.
In continuation of this observation, we
report Itere that M. leprae binas to the fol-
lowing phosphorylated proteins from the rat
peripheral nerve: a) a major 28-30-kDa
protein and h) a few minor proteins in the
' Received for publication on 14 Fcbruary 1997.
Acceptcd for publication on 31 Nlaurch 1997.
2 L. Ni. Suneetha. N1.Sc., Ph.D.; P. R. Satish, M.Sc.:
A. S. Balasuhrantanian, NI.Sc.. Ph.D., Neurochemistry
Laboratory, Christian Medical College Hospital, Vel-
lore, Tamil Nadu 632 004, Ilidia. ('. K. Job.
FR.C.Path., St. "rhonias Hospital and Lcprosy Centre,
Chettupattu, Muni! Nado 632 004, India. S. Suneetha,
M.R.R.S., D.C.l'., Schielíclin Lcprosy Research ^l
Training Centre, Karigiri, Tamil Nado 632 106, India.
Reprint rcyucsts to Prof. A. S. Balasubrantanian at
the above address or Fax 91-416-32035.
molecular weight range of 45-55 ki)a. For-
ther, we have characterired the 28-30-kDa
protein as a glycoprolein.
MA'I'1?RIALS ANI) ME'1'HOUS
"gris hydroxyntethyl antinontethane (Tris),
henrantidine hydrochloride, phenyl ntethyl
sulphonyl fluoride (PMSF) and sodiuni or-
thovanadate were obtained from Sigma
Chentical Contpany, St. Louis, Missouri,
U.S.A., and ganuua "P ATP was obtained
from the Bhaha Atontic Research Centre,
Bomhay, India. Other reagents used were of
analytical grade.
Rat peripheral nerve. Rats (3 months
old) were sacrificed hy cervical dislocation.
The sciatic nerves and brain were dissected
and further processe(' at 4°C. The tissue
was ntinced and homogenized in Tris HCI
huffer pH 7.6 (I O ntl/g tissue), containing
0.001% (w/v) benramidine hydrochloride,
200 iiM PMSF, 0.1 InM sodiunt ortho-
vanadate and 0.1% (v/v) Triton X-100. The
tissue homogenate was centmifuged nt
10,000 x g x 30 min in a Sorvai refrigerated
centrifuge. The supernatant was used for the
phosphorylation of proteins. Protein was de-
termine( according to Lowry, et al. ("). The
rat brain was isolated and processe(' as de-
scrihed for the peripheral nerve.
Protein phosphorylation. Protein phos-
phorylation conditions were as standardired
earlier in our lahoratory ("). The reaction
mixture contained 20 mM Tris-HCI huffer
pH 7.5, 20 ntM magnesium acetate and
gamma''P-Iabeled ATP (10 x 10' cpm) and
150 /tg of protein in 150 ,u1 of final volume.
The reaction was cardal out at 30°C for I O
min, at the end of which it was either sub-
jected to SDS-gel electrophoresis or cooled
on ice and used for hinding studies.
Bacilli. Al. leprae were isolated from
the skin hiopsies of horderline lepromatous
Ieprosy patients and from the foot pads of
352
65, 3^Suneetlia, et al.: M. leprae llinds to Nerre Gl.vcoprotein^353
inoculated mice and stored ai -20°C prior
to use as described earlier (''). The final
bacilli pellet was resuspended in 1 ml of 10
mIV1 phosphate buffered saline (PBS), pl
7.4, containing 0.1% bovine serum albumin
(USA). E.s.(1/erichici coli was obtained from
cultures provided by the Wellcome Re-
search Laboratory Institution. M.
vis was obtained from The Tuberculosis
Research Centre, Nladras. dulia. E. coli and
Al. bovis were heated ai 100"C for 2 inin
prior to use. Leproinin 11 was prepared as
described earlier (15). The storage ofM. lep-
me and lis treatinent with Na011, and heat-
ing of E. rol/ and Ai. /yori.s caused the inac-
lixado') of any protein kinases and protein
phosphatases in these organisms. M. /eprae,
lepromin H, Al. boris and E. coli, each of
10' bacilli, washed and suspended in PUS
containing 0.1(/e BSA. were used for bind-
ing studies.
Binding assay. The binding assay
methodology Wati hatiCCI upon earlier stud-
ies (".22). The bacilli suspende(' in 100-400
pl PBS containing 0.1% USA was added to
the -"p phosphorylated nerve proteins. This
was incubated ai 4°C with gentle shaking
for 2 hr. "I'() the suspension, 50 volumes of
buffer containing 145 mM NaCI, 10 InM
Tris HCI,^7.2-, and 0.1% (v/v) Triton X-
100 was added and microftwed fOr 1 min.
Under these conditions the bacilli were sed-
imented. The pellet was resuspended in
PBS and sedimented by centrifugation.
This step was repeated 7-10 times ai the
end of which 100 pl of SDS dissociado')
buffer 1186 'HM Tris HC1 bulfer, p11 6.8,
67 (v/v) beta-mercaptoethanol. 20% (v/v)
glycerol, 6% ( w/v ) sodium dodecyl sul-
fate and 0.001% (w/v) bromophenol bluel
was added to the penei and heated ai 100°C
for 3 min. Similar incubations were carried
out using Al. buris, E. coli and lepromin
SDS-gel electrophoresis was carried out on
12.5% gels according to Laemmli (").
ther processing and autoradiography of the
dried gels were carried out as mentioned
earlier
Concanavalin A-Sepharose comumn
chromatography of phosphorylated pro-
teins. Rat peripheral nerve proteins were
phosphorylated as mentioned earlier. The
phosphorylated proteins (400 lg,) were ap-
plied ou a column of concanavalin A-
Sepharose (0.5 x 3.0 cm) and the glycopro-
kDa
^1^2 3 4 5 6
70-
-
28 -3() -
IML I. Autoradiograin showing phosphorylateci
proteins: Lane 1 ShOWS l'-phosphorylated peripheral
nerve protein I I 5(1 pg) ( -P-PNI)). The same ;iniount
( 150 1.;) of was used for the hinding assay
with cach the 'mei'''. Lanes 2-5 show the `21)-1)NP
which are bound to the 'meteria ( 1(1 h2elIli used in
each case) after the binding assay. The binding assay
xv(ts carried out as described Ii Nlaterials anti Methods.
Bacilli used xveie: Lane 2 = lepromin II: Lane 3 = nil;
Lane 4 = E. coli: Lane 5 = boriv. Lane 6=1/. /eprae.
teins were eluted hv alpha methyl gluco-
side. The details oí the procedure are as
mentioned earder ('').
Periodic acid-Schilf staining for glyco-
protein. Staining fbr the gb,coproteins ou
gel aliei' SDS-gel electrophoresis of proteins
was done using the periodate-Schi ff reagent
according to Segrest and Jackson
RESULTS
An autoradiogram after SDS-gel elec-
trophoresis of the reaction mui mures in
xvhich rat peripheral 'terve extract was Meu-
bated with gamma '21) ATP ululei. the assay
conditions showed 32P-labeled proteins of
molecular weights ranging from 14-200
kDa (Fig. 1. lane 1).
The '2P-labeled rat nerve proteins were
ineubitted with Al. leprae. and the protein
bound to the bacteria was isolated by cen-
trifugation as deseribed in Materiais and
Methods. This Al. /eprae-protein complex
was subjected to SDS-gel electrophoresis
and autoradiography. The autoradiogram
showed a major 28-30-kDa protein and mi-
nor bands in the range of 45-55 kDa (Fig.
lane 6). indicating that these proteins
could bind to Al. leprae. Similar experi-
ments with At bori.s' (Fig. 1. lane 5) showed
relatively reduced levels of the 28-30-kDa
354^ International .1 ountal of. Leprosy^ 1997
kDa
-28-30
Fie.. 2 Autoradiograin showing -"P-phosphory-
lated 28-30-kDa protein eluted from the coneanavalin
A-Sepharose column, subjected to SDS-;_.tel elee-
trophoresis (12.5% gel).
protein and the other minor bands. E. coli
(Fig. 1, lane 4) and lepromin H (Fig. I, lane
2) showed only insigniticant leveis of the
bound 28-30-kDa protein. There were no
3213-labeled proteins in the control tube ia
which the binding assay was carried out
with the "P-labeled proteins in the absence
of bacilli (Fig. 1, lane 3).
Proteins of M. leprae, E. coli and M. bo-
vis did not undergo any phosphorylation as
observed in separate control experiments.
When the "P-labeled proteins of the rat
peripheral nerve were passed through the
concanavalin A-Sepharose column, the
28-30-kDa protein was found to bind to
the column. It could be eltaed with alpha
methyl glucoside (Fig. 2). The 28-30-kDa
protein also stained positive with periodate-
Schiff's staining on gel (result not shown).
Similar experiments were carried out
with the rat brain phosphorylated proteins.
None of the bacteria used in this study
showed any binding to the brain -"P-phos-
phorylated proteins (results not shown).
DISCUSSION
As mentioned earlier, M. leprae bind to
the extracellular matrix proteja FN in the
nasal mucosal cells and this, in turn, binds
to integrin FN receptors which help inter-
nalization of the bacilli (2. 18). However, this
mechanism does not explain the specilicity
of the bacilli for the nerve tissue.
To expiam the tissue specilicity of certain
pathogenic bacilli, another type of host
pathogen interaction lias beca described re-
cently wherein pathogenic bacteria bind to
specific manunalian cell membrane protein
receptors present on target cells. ,S7aphylo-
coccus atire tis is found to bine' to the 50-
kDa protein of endothelial cells (") and
Ui-et/pias/na urea/picum to the 30-k 1)a, 17-
kDa, 1 6-kDa and I 4-kDa proteins of human
epithelial cells (21)• These studies have
shown that the host cell membrane protein
reeeptors play a major role in tissue speci-
ficity of hacterial adhesion and invasion.
The 28-30-kDa protein is the major
phosphorylated glycoprotein of the rat pe-
ripheral nerve eluted from the concanavalin
A-Sepharose chromatography column by
alpha methyl glucoside. The molecular
weight and the phosphorylatable and glyco-
protein nature of the protein indieate that it
could be the P, protein of the peripheral
nerve (7)• P prCuein is the major myelin
protein of the peripheral nerve and lias a
signiticant role to play as a molecular Vel-
cro protein between myelin membranes
(3.21. Phosphorylated brama proteins did not
show binding to M. leprae, suLrgesting that
leprae lias specilicity to peripheral nerve
tissue protein.
The bindinL.,,,. of M. leprae to the major
28-30-kDa protein and a few minor 45-55-
kDa proteins was significantly higher com-
pared to that of /14. bovis and E. coli. This
may be due to the specificity of leprae ia
binding the protein. As observed ia our ear-
lier work, the decreased phosphorylation of
the 28-30-kDa protein in the presence of
leprae could be dile to the binding of M.
leprae to this protein (12).
The present work is focused on the phos-
phorylated host tissue protein binding to M.
leprae. Further experiments are needed to
identify the 28-30-kDa phosphoprotein and
the nature of the 45-55-kDa phosphory-
lated proteins which bind to M. leprae, and
to know whether any nonphosphorylated
proteins of peripheral nerve could bind to
leprae.
In conclusion, the present investigation
shows that intact M. leprae bind to a major
28-30-kDa protein and a few minor 45-55-
kDa proteins of the rat peripheral nerve
65, 3^Suneetha, et al.: M. leprae l3inds to Nerve Glycoprotein^355
phosphorylated in nitro. This M. leprae-pro-
tein interaction could have a role in patho-
genesis. The 28-30-kDa protein is the major
phosphorylatable glycoprotein of the rat pe-
ripheral nerve that resembles the P„ protein
of the peripheral nerve in its characteristics.
SUMMARY
To understand Mvcobacteriunt leprae-pe-
ripheral nerve interaction, we have investi-
gated the binding of M. leprae to rat periph-
eral nerve proteins in an in Nitro mudei us-
ina "P-phosphorylated proteins of the
peripheral nerve. Intact M. leprae binds to a
major phosphorylated protein of 28-30
kDa and, to a minor extent, to a few pro-
teins of molecular weight 45-55 kDa. This
binding was more specific for M. leprae
since only insignificant binding was ob-
served with other bacteria, such as M. bovis
or Escherichia coli. M. leprae did not show
binding to severa! phosphorylated proteins
of the rat brain. The 28-30-kDa binding
protein of the rat peripheral nerve was
found to be a glycoprotein by concanavalin
A-Sepharose column chromatography.
RESUMEN
Para entender la luteracción entre Mvcobacterium
leprae, y los nervios periféricos, investigamos el en-
latamiento in rimo de Al. leprae a proteínas fosfori-
ladas ("P) de nervios periféricos de la rata. El microor-
ganismo intacto se enlata a una proteína fosforilada de
28-30 kD y en major grado a algunas proteínas de
45-55 kD. El enlazamiento fuc relativamente especí-
fico para Al. leprae puesto que sólo se observó un en-
latamiento insignificante con otras bacterias como Al.
buris o Escheric/ia cali, M. leprae no mostró enlaza-
miento con varias proteínas fosforiladas dei cerebro de
la rata. La proteína enlazadora de 28-30 kD de los
nervios periféricos fue una glicoproteína que se podo
separar por cromatografía en colunma de seiarosa-con-
canavalina A.
RÉSUMÉ
Afin de comprendre I'interaction Mvcobacterium
leprae-nerf périphérique, nous avons analysé les liaisons
du M. leprae aux protéines des nerfs périphériques du rat
dans un modele in tino utilisant des protéines des nerfs
périphériques phosphorylées au -"P. Le M. leprae in-
tact se lie à une protéine majeure phosphorylée de
28-30 kDa et, dans une moindre mesure, à quelques
protéines de poids moléculaire 45-55 kDa. Cette liai-
son était plus spécifique polir A4. leprae, puisque seules
des liaisons non significatives ont été observées avec
d'autres bactéries, telles que Al. boris ou Esc/erichia
coli. Al. Leprae n'a pas montré qu'il se liait à dif-
férentes protéines phosphorylées du cerveau de rat. On
a troo vé par chromatographie en colonne sur con-
canavaline A-Sépharose que la protéine de liaisou de
28-20 kDa du nerf périphérique de rat était une glyco-
protéine.
Acknowledgntent. LMS and PRS are grateful to
the Colmei] of Seientifie & Industrial Research, New
Delhi, India, for awarding tfiem a research associate-
ship and senior research fellowship, respectively. The
work was supported by a grant from the Department of
Biotechnology (New Delhi). We are thankful to Mr.
Pugazenthi, St. Thomas Hospital, Chettupattu, for
technical help.
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M.leprae binds to a 28-30 kDa phosphorylated glycoprotein of rat peripheral nerve

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M.leprae binds to a 28-30 kDa phosphorylated glycoprotein of rat peripheral nerve

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