Relation of streptococcal M protein with human and rabbit tropomyosin: The complete amino acid sequence of human cardiac alpha tropomyosin, a highly conserved contractile protein

Vol. 142, No. 3, 1987 February 13, 1987

RELATION

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 813-818

OF STREP'TOCOCCAL M PROTEIN WITH HDMAN AND RABBIT

TROPOMYOSIN:

THE COMPLETE AMINO ACID SEQUENCE OF HJJMAN CARDIAC ALPHA TROPOMYOSIN, A HIGHLY CONSERVED CONTRACTILE Sheenah

M. Mische,

Belur

The Rockefeller Received

December

22,

N. Manjula,and

University,

PROTEIN Vincent

New York,

A. Fischetti 10021

N-Y.

1986

Partial sequences of group A streptococcal M proteins exhibit up to 50% sequence identity with segments of rabbit skeletal tropomyosin. It is well recognized that rheumatic fever and rheumatic heart disease in humans are sequelae of group A streptococcal infection. To examine whether the human cardiac tropomyosin would exhibit greater homology with the streptococcal M proteins, we have now determined its complete amino acid sequence. The amino acid sequence of human cardiac tropomyosin was established from sequence analyses of its peptides derived by cnzymic and chemical cleavages, and comparison of these sequences to the reported sequence of rabbit skeletal tropomyosin. These studies have revealed that the amino acid sequence of human cardiac alpha tropomyosin is identical to that of the rabbit skeletal alpha tropomyosin, but for a single conservative substitution of Arg/Lys at position 220. This observation increases the significance of the previously observed sequence homology between streptococcal M protein and rabbit skeletal tropomyosin and may have relevance to the pathogenesis of rheumatic fever. Furthermore, these results rank tropomyosin as one of the most highly conserved contractile proteins between 75 1987 Academic Press, Inc. vertebrate species reported thus far.

Group teria

A streptococcal

(l),

is

secondary

an alpha

(2-10).

hibit

up

50%

streptococcal

study

very

infection

cross-reactive

with

on the if

is

sera

homology muscle

determine

is of

streptococcal found

between

component, sequence this

analysis protein

is

the

with

regions

well

established

contractile

that

fever

skeletal

even

further

primary

heart

similarities

shown

alpha disease

contain

tropomyosin

and

tropomyosin been

heart

skeletal

the bacand to ex-

tropomyosin

complications

patients

and pathological

of the human cardiac exhibited

have

one of the

and mammalian and rabbit

of biological

proteins

and rheumatic fever

components M protein

rabbit

of

close

M proteins

of the

rheumatic

determinant

and exhibits

of streptococcal

rheumatic

acute

antiphagocytic

protein

with

sequences

It

the

coiled-coil

identity

(2,3,5).

Moreover,

a major

similarities

Partial

to

molecule

malian

helical

structural

myosin

sequence

M protein,

tissue. tropomyosin,

interest.

of

a

(11,12). antibodies Hence, a

the mam-

The present

was undertaken to with the M protein

molecule. METHODS powder

Human cardiac as described

tropomyosin was isolated from extracts (13-15). The protein migrated as alpha

813

of ether-dried muscle and beta components

0006-291X/87 $1.50 Copyright 0 1987 by Academic Press, Inc. AN rights of reproduction in any form reserved.

Vol. 142, No. 3, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(molar ratio 4.R:l) on SDS-PAGE. Attempts to separate these were unsuccessful. However, subsequent amino acid sequencing of human cardiac peptides showed no heterogeneity. This may be due to the beta component being at a much lower concentration or may suggest homology between alpha and beta human cardiac tropomyosin. Peptides of human cardiac tropomyosin for sequencing were obtained by cleavage with clostripain (7), trypsin (8), 2-nitro 5-thiocyanobenzoic acid (NTCB)(lG), CNBr and NBS (17). NTCB fragments were fractionated by gel filtration (16) and secondary cleavage by clostripain was performed. NBS peptides were separated on a equilibrated with 8M urea-0.05M tris-HCl-O.lM KCl, pH 7.5 QAE-Sephadex A25 column and eluted with a linear gradient of O.lM to O.3M KC1 in the same buff.er. All other peptides were purified by RP-HPLC on a Synchropak RP-P column (25 cm x 4.1 mm), using acetonitrile-TFA solvent system on a Waters HPLC unit equipped with an ISCO variable wavelength detector. Automated amino acid sequencing and C-terminal sequence analyses were performed as described (8).

RESULTS -ence -human ---.cardiac alpha ~Amino --acid -of ----data on alpha tropomyosin from human cardiac the

amino

data

acid

sequence

presented

in

COOH-terminal cardiac

tropomyosin

is

than

one type

presented

at position is

to be both It

tamic

for

cleavage resulted cleavage residues, also peptides

acid

of the human cardiac

the

at arginyl at most

arginyl

the

only

recovered

from

the (20).

for

be

derived

seen

from alpha

(18), residue

a total alpha from

more

the

data

tropomyosin

but

for

a single

of rabbit

skele-

This

detailed

is

study

of human

of present

may suggest

con-

(19).

substitution In the

observations

these

while peptides

addition

the

acid novel

clostripain 814

clostripain

digestion

resulting

exclusively

of the NTCB fragments to

resulting

glutamic for

157,

digestion

In peptides

third

The reason

bromide,

substitution

less

and

of the human

glu-

a study,

no

existence

tropomyosin.

clostripain

cleavages. residues,

notably

recovered. were

residues,

some unusual

but

The

human cnrdiac

the human protein.

reported

These

cleavage clostripain: Interestingly, -_ -by ------human cardiac alpha tropomyosin yielded in

lysine

conservative

at residue

alpha

for

with

(13).

cyanogen

tropomyosin

in

has been

cleavage

of human cardiac

alpha

in an independent,

was found.

muscle

of peptides may

sequence

along

and accounts

it

and genetically

acid,

difference

from

determined

position,

difference

of the 1,

degradations

trypsin,

sequence

skeletal

that,

an aspartic

of this

isoforms

intact

here

resulting

analyses

by an arginine

an additional

acid

evidence Unusual

substituted

Edman

Thus,

At this

a chemically

may be added

tropomyosins,

amino

rabbit

220.

skeletal

structure

molecule.

the the

rabbit

automated

clostripain,

by repeated

of the of

peptides

The final

1 that

by

and N-bromo-succinimide,

supported

to that

tropomyosin

sidered

of

well

in Fig.

substitution tal

acid

of digest

identical

of with

residues.

from

obtained

analyses

5-thiocyano-benzoic acid

tropomyosin

was

tropomyosin

of 284 amino

is

1

sequence alpha

2-nitro

of alpha

Fig.

tropomyosin: -A summary is shown in Fig.

muscle

residue

the from

expected cleavage

in a sequence is

not

of the from Nl and N2

peptides at glutamic of three,

cleavage

sites

clear,

cleavage

of the NTCB fragments.

but

from acid were these Novel

BIOCHEMICAL

Vol. 142, No. 3, 1987

AND BIOPHYSICAL

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Figure 1. Summary of the data establishing the complete amino acid sequence of alpha tropomyosin from human cardiac muscle. The amino acid sequence of rabbit skeletal alpha tropomyosin (18) is shown in the upper line in triple letter code. The amino acid sequence of human cardiac alpha tropomyosin, established from the sequence analyses of its peptides, is shown immediately below the rabbit tropomyosin sequence, with residues which are identical between the two species being indicated by dashes (-). As in the rabbit molecule, the N-terminal residue of the human cardiac tropomyosin has been found to be a blocked methionine residue. However, the nature of this blocking group in the human tropomyosin has not been established.

Peptide designations are based on the method of cleavage and are noted immediately under each fragment. Letters are used to designate the cleavage method: trypsin, T; clostripain, CP; NTCB, N; cyanogen bromide, Cb; NBS, NB. This is followed by a number which indicates the order of emergence of a particular peptide during its isolation by HPLC. When a secondary digestion was performed, the initial cleavage method is given first, followed by the second. The region of each of the human cardiac tropomyosin peptide sequenced by Edman degradation is depicted as forbeing represented by solid lines. -;fharrows ( 1, the remainder The residues were identified by carboxypeptidase digestion are indicated by back arrows The single difference in the human cardiac alpha tropomyosin molecule is a (-). at residue 220 (circled in human cardiacand boxed in rabbit LysfArg substitution skeletal alpha tropomyosin).

815

Vol.

142,

No. 3, 1987

cleavage

by

cleavage

BIOCHEMICAL

clostripain

have

at a glutamyl

been

residue

AND

BIOPHYSICAL

reported

has not

for

been

RESEARCH

lysine

COMMUNICATIONS

(7,21)

previously

and alanine

(22),

but

reported.

DISCUSSION Implications

--to streptococcal -and rheumatic heart

tococcus heart

damage

occurs

support

(11,12,23,24)

tients

with

is

obscure.

since

acute

disease: --disease is

demonstration

fever

tissue.

Subsequently,

streptococcal

antigen

that

with

demonstrated

the

hallmark

of the

demonstrated

that

cross-reactive

with

heart

rabbit

--et al (29) 5 streptococcus

have

disease.

More

antibodies

to

homology

between

skeletal

tropomyosin.

the

myosin

the

rod

former

region, (10).

tigen

of

cardiacthe

the

host

streptococcal some

of

the

the

as

the

streptococcal

components.

this

in

the

rabbit

Studies

cells

tial

for

regulatory

cal

component

systems.

properties

Tropomyosin

Non-muscle

of muscle

underway

of rheumatic

Evolutionary-significance: -in both muscle and nonmuscle contractile

are

pathogenesis

is

to

Cunto the

demonstrated

both

tropomyosin

significant than

study

human

amplifies segments

and

may

mammalian the

with

one an-

between

this

between

determine

and

streptococcus

more

(2-6),

that

than

the

identIty in

between

In muscle, effects share

and are

are

with

between

a ubiquitous

calcium-mediated

tropomyosin,

protein

of

explain

muscle

and

significance

of

fever.

(18,30-34). tropomyosins

have

observed

homology

observed

(28)

myosin.

have

more

tropomyosin

cross-reactions

the

have

The virtual

skeletal

have

we

demonstrated observed

(27)

antibody

one and may involve

the microbe.

previously

and the

being

relationship

a complex

a

of M5, M6 and M24 proteins

homology

latter

tropomyosin

immunological

homology

is

well

of

with

cross-reactive

antigens

as

M protein

sequences

the

homology

M5 and

(2,3,5)

that

bodies,

Aschoff

of a monoclonal

Subsequently,

the

skeletal-alpha

significance

partial exhibits

tissue

and rabbit

proteins

of Pep M5 protein Thus,

and the mammalian

streptococcal

of paextracts

consistently

and Beachey

the

studies

sera with

Murphy

the

Dale

which

has gained

tissue

and

in

strep-

demonstrated

muscle

membrane

Our earlier

the

reacted

Becker

cross-reaction

of rabbit

that (26)

mammalian

recently,

sarcolemmal

myosin.

(25) disease

proteins

sequence sequence

mechanism

studies,

and segments complete

of an autoimmune

with

significant the

by

and Meyeserian

other

demonstrated

with

the

mechanism

heart

contractile

human cardiac

ningham type

of

rheumatic

Kaplan

In

presence

between

the

by Cavelti

cross-reacts

the M protein.

association

and rheumatic

of human heart copurified

the

established,

The concept

the

rheumatic

Although well

protein

tropomyosin

is

an

on actin-based

many of the

implicated

and has been

physical

found essen-

regulatory and

in microfilament

chemi-

regulation

(31,32). The generalized nonpolar

sequence

of

sequence amino

acids,

tropomyosin

a-b-c-d-e-f-g and occupy

can be considered (35). the

In this "core" 816

a repeating model,

positions.

residues Those

heptapeptide "a"

and

at position

of the "d" "e"

are and

Vol.

142,

"g"

are

No.

usually

polar

or

charged,

ionic

acids,

In

the rabbit

pattern

represent

the

first

40 complete the

periods

nonpolar

stable All

within by

pomyosin Between

noncore all

but tropomyosin

pattern. the

it

interaction

with

feature

molecule

(19),

actin

(39). rabbit

Troponin

of the

present

dues tile

adjacent

sequenced

Contractile ple,

unlikely

has

to

alpha

proteins

are

exhibiting C is

considered

highly

demonstrating

change) (02. a 0.35% to date, tropomyosin is

2

with

a single between the most

the

11 conservative alpha

the

the

highly

cardiac

of

the

may be involved

in

assembly.

species

This

emphasizes

conservation

functional

The

heptapeptide

stability

but

tro-

(33).

human

constraints

more conserved

between

the

present

in

upon

the

or a 0.6% change substitution

and the human conserved

molecule

superfamily

species.

or a 1% change 1 residue difference

conservative rabbit

(38).

only

of 375 residues, molecules,

commonly

structure.

of homology

conserved, muscle

comtro-

pattern

filament

to be one of the

degree

most

repenting

diEferent

the

are

skeletal in

on

For

alpha

tropomyosin

the

periodicity

coiled-coil

out

in

thin

the

residue

a high

here

within

and reflects

helical

beta

molecule,

and charged

and the human skeletal study

influence

from

differences also

an

from

exhibits

tropomyosin

tropomyosins to date,

its

4

have

of the

position

the

(31-34).

heptad

rabbit

220 observed

of for

of homology

tropomyosins

differs

skeletal

"c"

degree

substitutions

from

rabbit

outer

molecules

of nonpolar

to maintain

of proteins

the

between

tropomyosins

with

alpha

tropomyosin

in

distribution

pattern

repeating

differ

at position

structure

difference

dominant

that,

is

tertiary

alpha which

identities

occupies

Hence,

single all

skeletal

heptad

conservative the

7

repeats

the basis

a high

tropomyosin

of

1 through resulting

in the forms

exhibit

and skeletal

are

hep-

(35,36).

repeating

beta

positions

substitution

coIled-coil

cardiac

residues

6

the

37 of which

residues molecule,

positions

molecule

repeating

arrangement

The regulririty and "d"

within

Thus,

are

the molecular

the

tropomyosin

to date

skeletal

noncore

the

positions

within

heptapeptide

tropomyosin

rabbit Rabbit

chicken show

arginine

"a"

sequenced

"e,f,g"

species,

pomyosin,

period.

the

The remaining molecule,

This of

COMMUNICATIONS

positions

"outer"

N-terminus.

short

of charge

residues,

its

"core"

isoforms

(37).

substitutions,

at

of

species, 39

in the

single

length

structure

identical

alpha

the entire

conservation

example,

found

molecule.

tropomyosin

and overall pletely

right of the

residues

coiled-coil

in

tropomyosin

heptad

at the

RESEARCH

positions.

found

skeletal

and a final

BIOPHYSICAL

"inner"

and are

begins

throughout

contiguously

AND

and occupy

amino

superstructure. tapeptide

BIOCHEMICAL

3, 1987

For

exam-

within species between the

(40).

The results in

tropomyosin among the

254

resi-

indicates contrac-

proteins.

ACKNOWLEDGMENTS* The authors would -----John B. Zabriskie for their encouragement Drs. Bruce A. Cunningham and James manuscript, Drs. D. M. Watterson and A. sions, and M. D. Simont for his invaluable manuscript. This work was supported by

like to thank Drs. Emil C. Gotschlich and and continued interest in these studies, M. Manning for critical review of the Seetharama Acharya for many helpful
817

Vol.

142,

No.

3, 1987

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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(HL25219 and AI11822 to VAF, and HL36025 to BNM). BNM was an Established Investigator of the American Heart Association during the tenure of this investigation. REFERENCES 1. Lancefield, RX. (1962) J. --Immunol. 89, 307-313 2. Iosein, B., McCarty, M.,and Fischetti, V.A. (1979) -Proc. Acad. Sci., --Nat. ___ U.S.A. 76/ 3765-3768 -3. Manjula, B.N. and Fischetti, V.A. (1980) J. Exp. Med. 151, 695-708 4. Phillips, G.N. Jr., Flicker, P.F., Cohen, Cx ManjulaTN.,nd Fischetti, V.A. (1981) -__Proc. Nat. Acad. Sci., 1J.S.A. 72, 4689-4693 5. Fischetti, V.A. and Manjula, B.N. (1982) In Sem. in Infec. Dis., Vol. IV: Bacterial Vaccines, Ed.,J.B. Robbins, J.C. Hill, and J.C. Sadoff (1982) Thieme-Stratton, Inc. N.Y. pp.411-418 6. !fanjula, B.N., Trus, B.L. and Fischetti, V.A. (1985) -Proc. Acad. sci . -Natl. .---USA. g, 1064-1068 7. Gjula, B.N., Mische, S.M. and Fischetti, V.A. (1983) -Proc. Natl. Acad. SCi ., ,o, 5475-5479 USA. 8. iYanjula, B.N., Acharya, A.S., Mische, S.M., Fairwell, T. and Fischetti, V.A. (1984) J. Biol. Chem. 259, 3686-3693 9. Iollingsheap.K,Fischetti, V.A. and Scott, J.R. (1986) -J. -Biol. Chem. --261. 1677-1686 LO. Manjula, and Fischetti, V.A. B.N. (1986) ____-Biochem. Biophys. Res. Commun., --__140, 684-690 11. McCarty, M. (1972) _In --_--Streptococci and ----Streptococcal Diseases, (Ed., L.W. Wannamaker and J.M. Matsen), Academic Press, Inc., New York, p.517 12. Zabriskie, J.B. (1967) Adv. in Immun. 1, 147-188 13. Cummings, P. and Perry, S.V. -___ (1973) Biochem: 2. 133, 765-777 14. Bailey, K. (1948) Biochem. J. 43_, 271-279 15. Hartshorne, D.J. and Mueller, H. (1969) --Biochim. Biophys. Acta 175, 3011-319 16. Ueno, H., Takahashi, S., and Ooi, T. (1977) J. Biochem. 32, 131-138 17. Ramachandran, L.K. and Witkop, B. (1969) % Met. Enz. vol. 11 Ed., C.H.W. Hirs and S-N. Timasheff, Academic Press, New?ork, pp.283-299 18. Stone D. and Smillie, (1978) J. -.Biol. L.B. --Chem. 3-53, 1137-1148 19. Dayhoff, M.O. (1978) Atlas of Protein Sequence and Structure Suppl. 5, Nat'1 --_-------Biomed. Res. Found., Washington, D.C. 20. Romero-Herrera, A.E., Nasser, S., and Lieska, N.G., (1982) Muscle ---me-.---- and Nerve 5 713-718 243 4683-4692 21. Mitchell, W.M., and Harrington, W.F., (1968) J. Biol. Chem. ---, 22. Chin, Anderson, P.M., and Wold, 7. (1983) --J. Biol. Chem. 258, C.C.Q., 276-282 23. Zabriskie, J.B. (1983) Phil. Trans. R- E-c-,,;o;~~ & 3p,3_, 177-187 24. Williams, R.C., Jr. (1983) Am. J. Med., -, 25. Cavelti, P.A. (1945) Proc. %&.Exp. Med. and Biol. 60, 376-381 26. Kaplan, M.H. and Meyeserian, -- M. (1962) LXet,,706-71527. Becker, C.G. and Murphy, G.E. (1969) --Am. J. ~Pathol. 55, l-37 28. Dale, J.B. and Beachey, E.H. (1985) J. Exp. Med., 162, 583-591 29. Cunningham, M.W., Hall, N. K., Krisher K.K. and Spanier, A.M. (1986) J2 munol. 12, 293-298 30. Smillie, L.B. (1979) ~-_Trends Biochem. Sci. 4, 151-155 _I 31. Cote, G.P. (1983) --mol. Cell. Biochem., 57, 127-146 (1985) --Cell and-Muscle Motility 6 141-184. 32. Payne M.and Rudnick, S.E. ~ -----9 -3 (1982) -Eur. 126, 293-297 33. Macleod, A.R. Biochem. -J. ---34. Ruiz-Opazo, N., Weinberger, J., and Nadal-Grinard, B., (1985) Nature 315, 67-70 Stewart, M., and Smillie, L.B. (1975) --J. Mol. Biol. 98, 35. McLachlan, A.D., 281-291 36. Crick, F.H.C. (1953) Acta Cryst, 6_, 689-697 37. Lewis, J. -Biol. W.G., and Smillie, --(1980)L.B. -Chem. 255, 6854-6859 (1980) -~J. Biol. Chem. 255, 38. Mak, A.S., Smillie, L.B., and Stewart, G.R. 3647-3655 (1979) Differentiation 14, 123-133 39. Vandekerckhove, J. and Weber, K. 40. Romero-Herrera, (1976) --Mol. Evol. 4, A-E., Castillo, O., and Lehmann, H.J. 251-270 818