BIOCHEMICAL

Vol. 86, No. 3, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

February 14, 1979

Pages 613-619

PARTIAL AMINO ACID SEQUENCE OF TWO FORMS OF HUMAN POST -YGLOBULIN C. TONNELLE, A. COLLE, M. FOUGEREAU, AND Y. MANUEL CENTRE D'IMMUNOLOGIE

INSERM-CNRS de Marseille-Luminy

Case 906 13288 - Marseille Received

October

25,

cedex

2 - (France)

1978

SUMMARY We have purified two different electrophoretic forms of Post-Y globulin defined by electrophoresis as "native slow" form and "fast" form respectively. Amino acid sequences of the first fifty-two residues of the "native slow" form and twenty-nine of the "fast" form were determined. The sequence shows that the "fast" form lacks the first nine amino acids of the "native slow" form. This observation is consistent with the existence of a "native slow" form that is degraded into other more acidic forms of Post-Y -globulin by loss of basic amino acids. INTRODUCTION Post12.000

daltons,

y-globulin was first

of cerebrospinal It

has also

urine, tic

fluid

forms,

variability showing were

after

antigenic

weight

storage

into

was

faster

become sequentially,

in humans as a constant

protein

component

tubular

disorders.

from patients of normal

(2,3,4).

storage, of urinary

amino

did

forms

forms, form,

any

and fast"forms, These

3 forms

but no diEference

of Py could

such that

then

(6).

serum, electrophore-

not observe

intermediate

acids

of about

: saliva,

in several

CEJKA (5),

Py . "Slow,

The various

electrophoretic the "slow"

humans

have been described

NH2 terminal

detected.

with

Py occurs

although

identity

shown to have different

lecular

protein

fluids

fluids,

in the mobility complete

weight"

and in urine

in several

and seminal

especially

"Low molecular

described (l),

been found

ascitic

( Py ),

be degraded

the slowest

the "intermediate"

form

in moupon could

and the "fast"

form. We have form which

is

previously

(6),

isolated

slightly

and which now on. The first results presented

and purified

slower

(more

a "fast"

cathodal)

will be refered of the sequence

than

form as well the

"slow"

as a new "slow" form

to below as the "native determination of these

described

slow" formfrom two forms are

and discussed.

Post y globulin : Py. Sodium Dodecylsulfate : S.D.S. High Performance chromatography

: HPLC. 0006-291X/79/030613-07$01.00/0 613

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved

Vol.

86,

No.

3, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

MATERIAL AND METHODS Preparation

of Py .

Py was obtained from urine of a patient with a congenital tubulopathy. NaN was used as preservative. As previously described (6), the P y was purl + ied through Biogel PI00 followed by ion exchange chromatography on DEAE-Sephadex A50 (Tris-HCl buffer 0.05M pH 8.8). The purity was determined by electrophoresis in 5% polyacrylamide-agarose gel (7), immunoelectrophoresis (8), isoelectric focusing (9) and 7% S.D.S. polyacrylamide gel electrophoresis (PAGE) (IO). Oxidation,

reduction

Disulfide bonds were oxidized with performic acid (11) or redudithiothreitol ; in the latter case, the half cystinyl residues were with iodoaeetamide (12).

ced with alkylated Amino

and alkylation.

acid

sequence

determination.

The total amino acid content was determined after 24h and 72h hydrolysis of native and oxidized protein in 6N HCl. The amino acid analysis was performed on a BECKMAN 121M analyser. Direct sequence analysis was obtained with the BECKMAN 89OC automatic sequencer using IM or O.lM Quadrol with polybrene, according to Klapper et al (13). PTH derivatives were identified by High Performance Liquid Chromatography (HPLC) according to the following conditions : a 5 minute exponential gradient of methanol-acetate (gradient curve no 10) was initiated with the analysis ( WATERS ASSOCIATES Liquid chromatograph ); initial concentrations : 25% methanol, 75% O.OlM sodium acetate pH 4.5. ; final concentrations : 45% methanol, 55% sodium acetate. The overall run time was 16 minutes. An aliquot of each PTH derivative was hydrolyzed with HI (14), and the corresponding amino acid residue was identified on the amino acid analyzer.

RESULTS Py was identified tested

for

monospecificity

preparation against

was tested total

red against nor against acrylamide da1 end. tive

form.

slightly we propose

by gel

human serum.

by isoelectric gel

anti-Py

described

inununoprecipitation, it

(6). using

was checked

anti lysozyme, antic sera. The Py preparation focusing,

serum, prepared and The purity of the Py a horse that

antiserum

no reaction

and anti showed

or polyacrylamide-agarose

occu-

A chains, one single or SDS poly-

electrophoresis.

The native The "fast" form The "slow faster

a rabbit

In addition,

anti@ 2microglobulin, anti-immunoglobulin

band revealed

via

as previously

than

to refer

form migrated in one single b&d at the most cathoappeared as a single band very distinct from the na-

and intermediate"

forms

appear

in fig.1,

at two positions

that described above as the "native" form. to the native Py as the "native slow" form.

614

Therefore

Vol.

86,

No.

BIOCHEMICAL

3, 1979

AND

BIOPHYSICAL

RESEARCH

C

I - Amino

acid

"intermediate" "slow" form

4-

"native

form

at 72h.

form

slow"

in 5W polyacrylamide-agarose

form

gel

of

composition. As shown in table

slow"

form

-

4-

Fig.1 - Electrophoresis the four forms of PY.

"fast"

COMMUNICATIONS

is a mean of three

The values

for

1 the amino acid

hydrolysis

results

Ser and Thr were

obtained

composition

of the "native

at 24h and two hydrolyses by extrapolation

to zero

time. The amino of the results corrections correct were

obtained (15)

for

acid

for

from three threonine

destruction

determined

amino

with

the following a - the

without

polybrene

resin.

from

sequence

The two forms tion,

24h hydrolyses.

acid

acid

acid

of the fifast"

and serine

during

as cysteic

2 - NH2 terminal

composition

hydrolysis.

In this

is

the average

case,

respectively The half

the oxidized

3% and 10% were made to

cystine

residues

protein.

determination.

of Py were

results "fast"

residues

form

subjected

to automatic

Edman degrada-

: form

The terminal

: 300 nmoles amino

acid

of oxidized

protein

was a Leucine

were used

and 24 resi-

dues were

identified (see table 2). b - the "native slow" form : 50 nmoles of reduced and carbo"native slow" Py were loaded into the sequencer, in the presence xymethylated of polybrene.

The first

residue

gave the PTH derivative, which eluted on HPLC but the back hydrolysis to free the amiat a position very close to tyrosine, no acid did not yield a tyrosine. Since in our system as in other systems

615

Vol. 86, No. 3, 1979

TABLE

: Amino acid mo1/10Omo1

I

Amino

BIOCHEMICAL

composition amino acid.

Acid

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

"native

the

PY "native

form"

slow"

and

"fast"

form

Py "fast"

of

Py

Difference

Lysine

6.26

5.43

-0.83

Histidine

2.71

2.52

-0.19

Arginine

6.22

4.56

-1.66

Aspartic

Acid

I I .o

Threonine

6.07

Serine

6.72

Glutamic

Acid

(1)

+0.53

“.53w 6.48

(1)

+0.39

6. 10c3)

10.42

-0.62

I I .63

+I .21

5.62

4.50

-I.

Glycine

7.32

6.75

-0.57

Alanine

8.90

9.38

Proline

Half

Cystine

2.8

(4)

I2

+0.48

3.64

(4)

+o.c34

Valine

7.85

7.92

+0.07

Methionine

3.38

2.93

-0.45

Isoleucine

I .02

I .63

+0.61

Leucine

6.71

7.11

+0.40

Tyrosine

2.92

3.14

+0.22

Phenylalanine

3.98

4.45

+0.47

(I) (2)

Values obtained 3% correction

(3)

10%

(4)

Analysis

(16.17)

correction

the

the

PTH

-

not

determined.

petitive

by

of

the

seryl

extrapolation

oxidized

residue

tyrosine,

of

to

residue

97%.

gives Ser

table

hydrolysis hydrolysis

(15). (15).

acid.

the

43

acid acid

a PTH derivative

is

3 onward,

(see

time. durizg during

: cysteic

occasionally that

zero

for destruction for destruction

derivative

we propose From

yield

linear

was made to correct was made to correct

that

NH -terminus.

Residue

2

positions

behaves

were

no

identified,

as 2 was

with

a re-

2).

DISCUSSION Comparison forms

of

by a simple

loss the NH2 terminal

NH2

terminal

The

"slow"

form

of

4 amino

acids.

the of

native

the

that

sequences

the

amino

acids

was

of

of

The

presence is (see

the'lnative

probably

form

"slow"

form

obtained

derived

from

the

of

an

arginyl that

3).

616

slow"

and

derived

from

form

Fy

from

"native

residue this

of

at starts

the

another

the

first

two

patient form

NH2 at

former

determined

and

slow"

"fast" the

We have previously

"intermediate"

suggestive table

is

peptide.

an

the

apparently

form protein

acid

of

latter

of an eight-residue amino

"intermediate" the

of

P ydemonstrated

by

(6). a loss

terminal residue

of no

8

Vol. 86, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

617

Vol. 86, No. 3, 1979

BIOCHEMICAL

TABLE 3 :NH2 terminal "native

slow"

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

amino acid sequence comparison

acids

Lru-Val-

for

the

increase

sensitivity dase, tion

Lys5,

forms,

normally

bed for

Pyin

be detected albumin,

ArgS. the loss

of this

protein in urine

data

of basic

further

Whatever evidence

for

ved from

the native

(18),

fluid, (6)

the different

acids

being

responsible

These

results

the present

the heterogeneity

of the various

has not

activity or bovine

enzymatic

results

forms

descri-

activity

substrates other

pepti-

was also

or esterase

to check

a high

the modifica-

an aminopeptidase

synthetic

the mechanism,

amino

explain

modification

no proteolytic

must be used

suggest

A contaminant

this

of

for

may, perhaps,

where

by a loss

may account amino

of P yusing

tests

characterized

mobility.

However,

Previously

in any sample

is

to proteolysis.

storage.

cerebrospinal

but

These

present

yet been described.

amino acid.

product

of electrophoretic

of the PY during

IO

Arg-

Each degradation amino

9

Lys-Pro-

(I) results obtained previously (6). (2) hypothetic position of NH2 terminal

electrophoretic

forms of Pv

4 6 7 a : (Sir)-(I~-Pro-Gly-L:s-Pro-Pro-Arg-Leu-Vat

form

"slow" form (I) "intermediate" form(l)(?) "fast" form

basic

of four

bring

of P ywhich

could serum

specifi@ities. unequivocal can be deri-

form.

ACKNOWLEDGMENTS It is a pleasure to thank Ms. MOINIER D., JARRY Th. and MOULIN A. for their skillful1 technical assistance during this work. assistance The authors gratefully acknowledge Dr. G. PICON for his clinical and the young Y.C. for donating urine. REFERENCES I - CLAUSEN, J. (1961)

Proc.

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170-172.

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