Vol.

172,

No.

November

BIOCHEMICAL

3, 1990

AND

BIOPHYSICAL

COMMUNICATIONS

RESEARCH

ISOLATION

1167-1174

Pages

15, 1990

AND CHARACTERIZATION OF A NOVEL PEPTIDE FROM PORCINE BRAIN Ken

Takamatsu*

Department

Kazuhiko

and

of Psychiatry

Stanford

Tatemoto

and Behavioral

University

School

Stanford.

AMIDE

of

Sciences

Medicine

CA 94305

Received Octcber 2, 199C

SUMMARY:

Peptide with C-terminal tyrosine amide was isolated from porcine brain by acid extraction and sequential steps of reverse amino acid and mass spectral anaiyses phase HPLC. Mierosequence, revealed the structure: Ac-Ala-Ser-Glu-Lys-Arg-Pro-Ser-Glu-ArgSince this peptide had the identical His-Gly-Ser-Lys-Tyr-amide. sequence to N-terminus of porcine myelin basic protein (pMBP) Iporcine myelin peptide amide 14 (pMPA14). 14, we have designated The final HPLC step yielded 20 ug of homogeneous peptide preparation from 20 kg brain tissue. Unilike other amidated peptides, pMPA14 may be produced by non enzymatic mechanism or unknown This unique amidation seems to occur examidating enzyme. ‘C’ 1990Acadeinlc Press,1°C. clusively to MBP in the brain.

Many naturally tides

contain

structure

that and

of

converted

and identified cal

C-terminal

detection possess intestine

the

into

C-terminus

isolation discuss

by using

peptide

is

technique, the

active

the

off

derivative,

a search

was carried

extracted

for

out

porcine

and a number

and characterization

brain of

of generation

Using

previously

amide structure

were isolated(3,4,5,6,7). of curious peptide having

the hypothesis

pep-

by a proteolytic

chromatography(Z).

C-terminal

in

secretary

Peptides with such a technique in which chemical

cleaved

the dansyl

by thin-layer

hormonal peptides high concentrations its

biologically

amide structure(l).

can be assayed

the C-terminus zyme,

occurring

extract. this

selectively this

chemi-

unknown peptides

in extracts of

en-

novel Recently tyrosine

of

brain

neural

we found amide at

We now describe novel

peptide

mechanism of

this

and

amide

the and

peptide.

* Present Address: Department of Physiology, Keio University School of Medicine, Shinjuku, Tokyo 160, Japan. Author to whom correspondence should be addressed.

Vol.

172, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

METHODS Tissue Extraction: Porcine brain (20 kg) was boiled in distilled water for 20 min, cooled and then homogenized. Acetic acid was added to 0.5 M and the homogenate stored at 4°C overnight with stirring. The peptides in the extract were adsorbed onto alginic acid at pH 2.7, eluted with 0.2 M HCl and lyophilized. Chromatograuhic Purification: The lyophi lizate were dissolved with 0.2 M acetic acid and loaded onto Sephadex G25F (10 x 150 cm), then eluted with the same buffer. Fractions of Mr l-3K were loaded directly onto reversed phase MCIgel ODS-1OU column (20 x 250 mm, Mitsubishi), then eluted with a linear gradient of acetonitrile in 0.1% TFA at a flow rate of 10 ml/min. Fractions were collected at 1 min intervals and assayed for the chemical detection of a C-terminal amide. Fractions containing tyrosine amide were then loaded onto reversed phase TSKgel ODS-120T column (4.6 x 250 mm, Tosoh), and developed at a flow rate of 1.0 ml/min with a linear gradient of acetonitrile in 5 mM phosphate buffer pH 6.5. Fractions from second HPLC containing tyrosine amide were loaded onto reversed phase Chemcosorb 5-ODS-H column (4.6 x 250 with a linear gradient of acetonitrile in mm. Chemco) , eluted 0.1% TFA at a flow rate of 1.0 ml/min. Each peak were collected and assayed for tyrosine amide. Chemical Assay of pMP4: Porcine MPA was assayed by the chemical method based on the detection of a C-terminal tyrosine amide which was released by the treatment of sample with thermolysin or trypsini2). Fragmentation-of pMPA: Approximately 5 nmole of intact peptide were digested with lysyl endopeptidase (Wake) or thermolysin (Boehringer) in 10 ul of appropriate buffer for 6 hours at 37”C, and were directly fractionated on Chemcosorb 5-ODS-H column (4.6 x 250 mm) using a linear gradient over 45 min of 0-15X acetonitrile in 0.1% TFA at flow rate of 1.0 ml/min. Amino Acid Analysis: Approximately 100 pmoles of purified peptide was hydrolysed by vapor HCl at 1lO’C for 20 hours, and was applied to automated amino acid analyzer (Japan Spectroscopic Co.). Amino Acid Sequence Analysis: Samples of purified peptide or its proteolytic fragments were subjected to automated Edman degradation on Applied Biosystems A-470 gas phase sequencer, and phenylthiohydantoin derivatives of amino acids were analyzed by HPLC as described (8). Mass spectral Analysis: Positive ion spectra of intact peptide was obtained using JEOL JMS-AX505H FAB mass spectrometer. The mass value for the intact peptide is the monoisotopic mass of the protonated molecular ion. Peptide Synthesis: Peptides were sysnthesized manually using the method of Fmoc-polyamide synthesis(9). Approximately 10 nmoles Exposure of Peptides to Oxypen Radicals: of synthetic peptide, Lys-Tyr-Leu, was dissolved with 20 ul of 20 mM sodium phosphate buffer pH 7.0 followed by addition of cupric chloride (l-100 uM) or ferric chloride (0.1-5 mM), ascorbic acid (l-10 mM) and hydrogen peroxide (0.1-5 mM). Where appropriate chelator or radical scavenger was added before the cupric or ferric chloride. Samples were incubated at 37°C for 1 hour and then passed through Sep-Pak C18. The formation of C-terminal amide was examined by using the chemical method (2). RESULTS Purification G25F were

of pMPA: The fractions subjected to reversed 1168

of Mr l-3K from the phase HPLC on MCIgel

Sephadex ODS-1OU

Vol.

172,

No.

BIOCHEMICAL

3, 1990

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

b

Fin.1. Chromatographic isolation of MPA from porcine brain. (a) First reversed phase HPLC profile of the fraction of Mr l-3k from Sephadex G25F chromatography. An aliquot (200mg) of the fraction was applied to MCIgel ODS-1OU (20 x 250 mm) and eluted at a flow rate of 10 ml/min with a linear gradient of acetonitrile in 0.1% TFA. The fractions containing tyrosine amide (coated area, Yl and Y2) were detected by using the chemical assay method. Y2 corYl was further purified by the responded to neuropeptide Y. Then, successive reversed phase HPLC on TSKgel ODS-12OT (4.6 x 250 mm) with a linear gradient of acetonitrile in 5 mM sodium phosphate pH 6.5 at a flow rate of 1.0 ml/min and on Chemcosorb 5-ODS-H (4.6 x 250 mm) with a linear gradient of acetonitrile in 0.1% TFA at a flow rate of 1.0 ml/min (b).

(Fig.l-a).

Two

detection

method.

corresponded (Yl) tides (5). phase

tyrosine

to

had

The

peak

HPLC

amide was

on

TSKgel

Table

1. Amino

Amino

acid

peaks

Total no. residues N-terminus C-terminus

peak

Y

(NPY)

profile (peptide

further ODS-120T,

and

acid compositions its proteolytic No.

1.1

(1)

2.0 2.1 1.0 1.1 1.8 0.9 2.8 1.1

(2) (2) (1) (1) 12) (1) (3) (1)

found (Y2)

early

from

those

of

the

1.0 0

porcine

the

(If (0)

0.9

(1) 0 (0) 0 (0) 0.9 (1) 0 (1) 0.9 (1) 0 (0) 4

Blocked

Blocked

peak

known

pep-

polypeptide) reversed

5-ODS-H

MPA and

Th2

14

1169

amide

eluting

1.0 0 0 0 0 0 0 0 0

(1) (0) (0) (0) (0) (0) (0) (0) (0)

of

Tyr-amide

chemical

successive

Chemcosorb

residues Ll

the

tyrosine

panceratic

of the fragments of

of

The

by on

by

(3). YY,

purified

pMPA14 Ala h-g Glx GUY His LYS Pro Ser TYr

were

eluting

elution

tyrosine

This

late

neuropeptide

a different

with

amide

1

(Fig.l-

Vol.

172,

No.

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

a

b

-

1:11:, 3’.. ,

0

40

Elution

Volume

50

(ml)

Fig.2. HPLC separation of lysyl endopeptidic (a) and thermolytic (b) fragments of pMPA14. Approximately 5 nmoles of pMPA14 were digested with lysyl endopeptidase or thermolysin in 10 ul of appropriate buffer for 6 hours at 37°C. After adding 100 ul of 0.1% TFA, the enzyme digests were applied to Chemcosorb 5-ODS-H (4.6 x 250 mm) and eluted at a flow rate of 1.0 ml/min with a linear gradient of O-15% acetonitrile in 0.1% TFA over 45 min.

The

b).

peak

homogeneous

and

Structural 14

intact

amino

peptide

had thermolysin

HPLC

(Fig.2-a,

blocked

pMPA14

PTH

amino three

five

fragments

1).

which

of that

of

the and

were acid the

eluted

1170

in of

blocked

N-

TM

ASOKFlPSQR~HSKYLASAS?---

Fig.3. Complete amino acid sequence dopeptidic fragments and Thl-5 are t i f ied by sequence and amino acid amide was identified by the elution chemical assay method.

by

analysis

L2 -___ TM

Th3

this

treatment

separated was

the

peptides

-NH2

~

AC-

amino

indicating

L3

-7 Th2

pMBP

analysis

fragments

ASOKRPSORHGSKY L1

be

consisted

indicated

acids,

The

(Table

to purified

amide

fragment,

Acetylalanine.

AC-

highly

Sequential

thermolytic

alanine

this

tyrosine

Treatment

yielded Th-2, to

only

of with

I).

found

analysis.

analysis

N-terminus.

yielded

position yielded

no

was

structural

(Table

endopeptidase b).

amide,

peptide

residues

the

with identical

acid the

generated

lysyl

to

Amino that

acid

peptide

tyr-osine

subjected

suggested

with

Th-2

was

Studies:

material of

contained

of pMPA14. Ll-3 are lysyl enthermolytic fragments idenanalyses. C-terminal tyrosine profile of HPLC and by the

Vol.

172,

No.

3, 1990

Table

BIOCHEMICAL

AND

Inhibition

of

2.

Control EDTA Histidine

Control phosphate ascorbic

the

be

and

The

added

from

of

by

20 brain

chemical was

reducing

agents

of

the

ug

13

amino

gave

be

as

in

a value

protonated

of

the

results, to

of

molecular

homogeneous system

peptide

using

Copper

The

or

amount

was

such

as

superoxide

Lys-Tyr-

ferric

radicals

ions

capable

estimated

ion-dependent

of to

reactions

ascorbate,

mannitol, by

reveal these

peptide

hydroxyl

ferric

chelators,

to

of

tissue.

2).

or

analysis

deduced

amidation

generated

copper

sodium 2 mM

From was

examined.

(Table

inhibited

able

intact mass

peroxide

copper

mM

sequence

peptide

porcine

The

The

slightly

kg

amide

pmole/hour.

only

this

yielded

substrate

hydrogen

stimulated

steps

20

peptide

by

0 35 100 46 35 30

were

monoisotopic

PMPA:

a model

hibited

mM mM

analysis

HPLC

of to

mM

0.5 1.0

100

acetylalanine. of

the

final

forming 45

N-terminus,

for

preparation as

0.1

fragments

spectral

Generation

mM mM mM mM

N-terminal

structure

Mass

ion.

Leu

acetylalanine.

excluding

(M+l)

(%)

80 52 55 55 60 80

unit/ml 100 unit/ml 100 unit/ml 1.0 mg/ml

thermolytic

primary

1670

Inhibition

was incubated in the solution of 20 buffer pH 7.0, 2 uM cupric chloride, acid and 1 mM hydrogen peroxide.

to

Fig.3.

amidation

mM mM

0.5 1.0 100 100

Manni to1 Catalase Boiled catalase Superoxide dismutase Bovine serum albumin cu (-) Cu C-j, Fe (+)

acids,

COMMUNICATIONS

0

1.0 0.5 1.0

Imidazole

tryptic

chemical

RESEARCH

Concentration

Inhibitor

terminus

BIOPHYSICAL

markedly

thiourea,

be

were in-

catalase,

and

dismutase.

DISCUSSION The porcine

C-terminal brain

structure ment

this

porcine

normal

MBP

amide.

In

molecular

extract

of of

tyrosine

amide basic

this with

agreement of

the

peptide

chemically

peptide myelin

fragment, weight

and

amide

was

isolated

characterized. corresponded

protein

(MBP)

from The

to 1-14.

the

primary

N-terminal

frag-

However,

unlike

peptide contains a unique C-terminal the proposed structure, the chemical intact

peptide 1171

was

found

to

be

1669

dal-

Vol.

172, No. 3, 1990

tons

BIOCHEMICAL

(calculated

peptide

1669.4).

of

nervous ing

from other

tive

peptides.

since brain

C-terminal zymes,

we could proteins

Since

for

the

of

is

C-terminal

of

be a leucine, be responsible

of

pMPA14,

to Ser-Lys-Tyr-amide

either

hitherto

pMPA14

(data for

en-

mandatory (10,11,12).

predicted

from

respectively, the for this arnidation.

not

amidating

may be responsible

the

residue

the C-terminal

unknown

neuroac-

by amidating

using a partially failed to demonstrate

Ser-Lys-Tyr-Leu,

mechanisms

residue

originat-

of

is

glycine

would

synthetic

in central

amides

catalyzed

enzymes may not

of that

occurs

peptide

specificity

extended

our experiments amidating enzyme

which

porcine myelin from MBP. The

MBP and precursors

usually

extended

MBP structure,

results pituitary

find

substrate

C-terminal

amidating

event

not than

amidation

a characteristic

requirement the

we have designated MPA might be generated

MPA may be a unique

system,

RESEARCH COMMUNICATIONS

Then,

amide 14 (pMPA14).

production

AND BIOPHYSICAL

the

known The

purified bovine the conversion extended

shown).

fragments

This

indicated

enzymes

or non-enzymatic

C-terminal

amidation

of

MPA. Non-enzymatic demonstrated amidating

amidation

using system

in is

vitro

of

peptides

models

dependent

(13).

was This

on transition

previously

non-enzymatic

metals

such as cop-

and molecular oxygen and shows the participation per, ascorbate of hydroxyl and carbon radicals as reaction intermediates in production and

of

ferric

a peptide

ions

amide.

generated

Lys-Tyr-amide

presence of hydrogen peroxide. such as rnannitol appeared to suggested

that

Fenton-type 01

iron

further indeed

MPA might

reaction.

containing studies occurs

Since

under

is required in the brain

revealed from in

radical this

by hydroxyl

such as these physiological

that

Lys-Tyr-Leu

hydroxyl

influence

be produced

Reaction fluids

Our results

the

copper in

the

scavenger

reaction,

it

radicals

via

is

may occur

in copper

condition.

However,

a

to determine whether such a reaclion and contributes to oxygen radical

damage of myel in sheaths. It

has been reported

experimental

allergic

that

N-termianl

encephalomyelitis

fragments of MBP induce (EAE) in susceptible

mouse strain, PL/J and BlO PL mice (14,15). Precise experiments showed that N-terminal 6 residues constitute the antigenic core which is essential as the recognition site (16.17). MHC Class II the N-terminal strucrestricted helper T-lymphocytes recognize ture

via

identical

their antigen length of

specific receptors (18,19). amino acid sequence with 1172

MPA had an these en-

Vol.

172,

No.

cephalitogenic terminal

BIOCHEMICAL

3, 1990

peptides. amide

AND

Furthermore,

structure.

These

that MPA act as an autoimmunogen sociation with limited class II some

pathological

time

we do not

MPA in

brain

MPA in

central

or have nervous

results

reliable

COMMUNICATIONS

MPA contained

a unique

suggested

conditions. method

for

The physiological system

RESEARCH

the

remain

and

At the

the

asin

present

quantitation

pathological

C-

possibility

or immunological tolerant in and T-ccl 1 receptor molecules

physiological

the

and CSF.

BIOPHYSICAL

of roles

of

to be determined.

ACKNOWLEDGMENTS The authors wish Makk and Mrs. P.Angwin. formed by the Center School of Medicine.

to acknowledge the assistance Mass spectral determinations for Mass Spectrometry, Keio

of Mr. G. were perUniversity

REFERENCES I .

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16.

Narita, K., Matsuo, H., and Nakajima, T. (1975) In Protein Sequence Determination (S.B. Needlemann, Ed.) pp.30-130. Springer, Berlin. Tatemoto, K., and Mutt, V. 11978) Proc. Natl. Acad. Sci. USA 75, 4115-4119. Tatemoto, K., Carlquist, M., and Mutt, V. (1982) Nature 296, 659-660. Tatemoto, K., and Mutt, V. (1981) Proc. Natl. Acad. Sci. USA 78, 6603-6607. Tatemoto, K. (1982) Proc. Natl. Acad. Sci. USA 79, 25142518. Tatemoto, K., Rokaeus, A., Jornvall, H., McDonald, T.J., and Mutt, V. (1983) FEBS lett. 164, 124-128. Tatemoto, K., Efendic, S., Mutt, V., Makk, G., Feistner, G-J., and Barchas, J.D. (1986) Nature 324, 476-478. Hawke, D., Yuan, P.M., and Shively, J.E. (1982) Anal. Biochem. 120, 302-311. Atherton, E., Caviezel, M., Fox, H., Harkiss, D., Over, H., and Sheppard R.C. (1983) J. Chem. Sot. Per-kin Trans. 1, 65-73. Bradbury, A.F., Finnie, M.D.A., and Smyth, D.G. (1982) Nature 298, 686-688. Kizer, J.S., Busby, W.H., Cattle, C., and Youngblood,W.W. (1984) Proc. Natl. Acad. Sci. USA 81, 3228-3232. Kizer, J.S., Bateman, R.C., Miller, C.R., Humm, J., Busby, W.H., and Youngblood, W.W. (1986) Endocrinol. 118, 2262-2267. Bateman, R.C., Youngblood, W.W., Busby, W.H., and Kizer, J.S. (1985) J. Biol. Chem. 260, 9088-9091. Zamvil, S., Nelson, P., Trotter, J., Mitchell, D., Knobler, R., Fritz, R., and Steinman, L. (1985) Nature 317, 355-358. Zamvil, S., Mitchell, D.J., Moore, A.C., Kitamura, K., and Steinman, L. (1986) Nature 324, 258-260. Wraith, D.C., Smilek, D.E., Mitchell, D.J., Steinman, L., and McDevitt, H.O. (1989) Cell 59, 247-255. 1173

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172, No. 3, 1990

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

17. Urban, J.L., Horvath, S.J., and Hood, L. (1989) Cell 59, 257-271. 18. Zamvil, S.S., Mitchel, D.J., Lee, N.E., Moore, A.C., Waldor, M.K., Sakai, K., Rothband, J.B., McDevitt, H.O., H. (1988) J.Exp.Med. Steinman, L., and Acha-Orbea, 167, 1586-1596. 19. Acha-Orbea, H., Mitchel, D.J., Timmerman, L., Wraith, D.C., Tausch, G.S., Waldor, M.K., Zamvil, S.S., McDevitt, H.O., and Steinman, L. (1988) Cell 54, 263-273.

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