Interactions of an ovalbumin glycopeptide with concanavalin A

Vol. 90, No. 1, 1979 September

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

12, 1979

Pages

INTERACTIONS

117-122

OF AN OVALBUMIN GLYCOPEPTIDE WITH CONCANAVALIN A C.F.

Brewer

Departments of Molecular Pharmacology and Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, N. Y. 10461 Received

July

23,1979 SUMMARY

The interaction of a highly purified glycopeptide isolated from ovalbumin with Concanavalin A has been investigated by measuring solvent proton relaxation rates over a wide range of magnetic fields. We find that binding of the glycopeptide to Mn-Ca-Concanavalin A uniformly reduces the solvent proton relaxation rates in the same manner as that of simple saccharides such as methyl a-D-mannopyranoside, but that the magnitude of the reduction is not as great. Furthermore, we observe that the glycopeptide is capable of precipitating the lectin, and that the precipitation reaction can be readily reversed by addition of methyl a-Dmannopyranoside. The latter results indicate that the branched chain glycopeptide appears to be bivalent with respect to binding by the lectin.

to its cells

Interest

in

capacity

to bind

(1).

binding

the jackbean

The specificity

properties

to be directed investigating of relating

with

cell

Larmor Sl site,

of the toward

surface rates,

surface

membranes

protein

the monosaccharides

glucose

and saccharide

the molecular

over

frequencies

a wide

from

0.01

binding

of simple

saccharides

nuclear

magnetic

of mono-

and oligosaccharides

a single

residue

binding

oligosaccharides

were

nearly

for

A.

of Mn-Ca-Con reduced

Con A, such as the a(l+Z)

solvent

the

A (Mn

we studied

greater

mannans

2+

in the

upon

technique,

that

proton

to proton

by &20%

this

We concluded and that

per monomer,

the

the

to interact

(corresponding

Using

been

ability

that

(NMRD) measurements,

to Mn-Ca-Con site

(3).

(2)

of Con A, with

to its

fields

et al.,

We have

properties

uniformly

to the protein disperison

to the saccharide

we demonstrated

of magnetic

and transformed

and mannose.

to 50 MHz) of solutions

the 52 site),

resonance

range

normal

related

of the lectin

Recently

carbohydrates.

due primarily

shown by Goldstein

binding

properties

A) is

of both is

has been

ion

A(Con

interactions

which

Ca2+ in

certain

Concanavalin

of these

the metal

goal

relaxation

to cell

lectin,

called the binding

the protein affinity relative

possesses of

to

the

0006-291X/79/170117-06$01.00/0 117

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

Vol. 90, No. 1, 1979

corresponding binding

BIOCHEMICAL

monosaccharide,

associated

with

AND BIOPHYSICAL

was due to an increase

the presence

of more

than

RESEARCH COMMUNICATIONS

in their

probability

one saccharide

of

binding

residue

in the molecules. This action

present

communication

of a glycopeptide

monitored

isolated

by NMRD measurements.

the glycopeptide first

describes

is

observation

capable

our initial

observations

from

ovalbumin

(Fig.

In the

course

of these

of precipitating

of such a reaction

Mn-Ca-Con

studies,

the lectin.

between

MATERIALS

1) with

on the

This

interA as

we observed appears

Con A and an isolated

that

to be the

glycopeptide.

AND METHODS

Con A was obtained from Miles-Yeda and demetallized to give apo-Con A as previously described (4). Mn2+ and Ca2+ were added as their chloride salts to solutions of apo-Con A by addition with a microliter syringe, and enough time was allowed from the protein to be converted to the active, "locked" form (4). Protein concentration was determined spectrophotometrically at pH 5.6 using an absorbance Methyl a-D-mannopyranoside was obtained from Pfanstiehl Labo$g$=;: 12.4 (5). The ovalbumin glycopeptide was a gift from Dr. Paul Atkinson; its purity was determined to be greater than 90% as determined by 'H nmr at 360 MHz (Carver, J. and Atkinson, P., unpublished results). NMRD measurements were made at the IBM T.J. Watson Laboratories, Yorktown Heights, N.Y. Details of the technique and theory can be found elsewhere (6) RESULTS AND DISCUSSION The top absence terms sion

data

in Fig.

of saccharide

that

has been previously

that

a water

solvent tion

in

of the residence

observed rates

profile.

under

therefore Addition

the binding rates

at all

relaxation (TV)

of the

small'

conditions compared

of sufficient sites fields

is

are

exchanging

amounts

2,(X)),

fairly

are

rapidly

There

is

exchanging

water

molecule(s)

of methyl in

118

of and

bulk contributo the

to the

(f or example,

observed

apo-Con

A) and

contribution. a-D-mannopyranoside

a nearly

as we previously

all

a significant

of the protein

1 set -l

that

with

in

disper-

to the protein,

profile.

than

result

relaxation

features bound

A in the

of the profile

of magnetic

tightly

to the paragmagnetic

of the protein (Fig.

theory

contribution less

analysis

The important

A complex

The diamagnetic

these

the

(6).

of the Mn 2+ is

time

of Mn-Ca-Con

A quantitative

into

published

the observed

the NMRD profile

25'.

enter

the Mn-Ca-Con

ligand(s)

to give

2 shows

at pH 5.6,

of the parameters

the Mn2+ ions

is

(0)

uniform reported

reduction (3,6).

to saturate in Analysis

the

Vol. 90, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

. MAN

MAN

al.2

l

MAN

.

.

a13\MANp

I MAN

1.6

. .

l

.

+

+

+

+ .

adMA~4

+

. 0.

P 1.4

+

+ +

+

xx

I

+ x

GlcyAc

x x

+

+

x

x

x xx

x

I

Bl.4

GlcNAc

0

1

I

02 0.01

I ASN

Fig. 1. indicates

Glycopeptide isolated N-acetyl-glucosaorine;

I

0.1

/

1.0 FREQUENCY

from ovalbumin. Man indicates Asn indicates asparagine.

-i

10

100

(MHz)

mannose; GlcNAc

Fig. 2. NMRD spectrum of a pH 5.60 solution (buffered with 0.1 N potassium acetate, 3.9 M potassium chloride) containing 0.46 mM Mn-Ca-Con A (0); the same solution in the presence of 1.82 mM methyl a-D-mannopyranoside (+); and the protein solution in the presence of 1.7 mM ovalbumin glycopeptide (X). The Mn-Ca-Con A was prepared by adding 0.46 mM Mn Cl2 and 1.3 mM CaC12 to a solution of 0.55 mM apo-Con A and allowing the solution to stand until Mn-CaCon A formation was complete. Under these conditions, 0.09 mM Ca-Ca-Con A was also formed. The temperature for all of the measurements was 25'.

indicates the

that

the most

reduction

of the profile

of the Mnzf. thus

ride

in

appears

the presence

and not

the monosaccharides relaxation that

rate

is

the effect

NMRD profile We have

as considering

hindrance

sensitive

to the

affinity

other

that

data

shown

the effects

that

in the

that

119

the

change

of the

a variety

is This

profile

for

from

these most

saccha-

ligand(s) in

by

the observed used,

and thus the

of mono-

all

the

and

(3).

We

of the

results likely

in

lectin.

of the protein

the protein

to

ligand(s) ion

saccharide

with

to Con A, and we suggested literature

water

interaction

dispersion

water

in Con A upon

bound,

of adding

rise

to the protein.

of saccharide

A on the NMRD of solutions

in the

give

on the metal

bound

constants

of saccharide

same change bind

ligands

transition

to the amount

investigated

the

water

of the exchanging

further

monitor

which

in rM of the exchanging

of a conformational

We have

to Mn-Ca-Con

tested

the parameters

monosaccharide

(3,6).

recently

essentially

saccharides

of this

to steric

a sensitive

oligosaccharides observed

an increase

was proportional is

in

of the exchanging

to be a result

binding,

change

is

The lifetime

increased

change

significant

as well possesses

Vol. 90, No. 1, 1979

a single

residue

such as the more than

binding

a(l+2)

mannose

of their

In this

carbohydrate

involve

a similar

residues

were

apparent

affinities have

8).

i-

in

found

for

amounts

of methyl

reduced

the observed

to the protein, displaced

simple

from

the

to check

rates

were

possesses

molecules

of

these

relative

to

the interactions

1) from

of

of

but

It

that

the

similar

is

Addition

glycopeptide-Mn-Ca-Con

therefore, of the protein not

could

as great

of large A solution

of the monosaccharide

the glycopeptide

to however,

appears,

increase

(3).

in

was reduced

on the Mn 2+ ion

characteristic that

was added

to the protein,

(~40%).

ligand(s)

to the

ovalbumin

NMRD profile

and oligosaccharides

experiments for

mM while of a stock

unchanged. same rates,

were

concentration

The glycopeptide

the

the range

binding

be competitively

lectin.

Con A complex

by addition

also

or glucose

for

was qualitatively

as great

complex,

to those

might

constants

was added

water

demonstrating

of dilution

mM, and too.28

(Fig.

reduction

was not

mono-

rates

A series

NMRD data.

This

exchanging

more

NMRD techniques.

a-D-mannopyranoside

thus

to examine

a-D-mannopyranoside

reduction

with

mannose

chain

begun

A, the observed

2(+).

lectin

to account

branched

glycopeptide

the glycoprotein

as that

begin

affinity

Con A using

in Fig.

of the

could

multiple

the

on the protein.

and glycolipids

where

of

increases

site

of the

enhanced

thus

of Mn-Ca-Con

of the

increased

cases

which

binding

interaction

Con A that

We have

when methyl

the lifetime

the

in

as their

purified

as shown

the magnitude

for

with

to a solution

that

mechanism

as well

(cf.

molecules,

to an extended

that

oligosaccharides

to be due to the presence

such as glycoproteins

constants

exhibited

observed

and not

RESEARCH COMMUNICATIONS

of certain

in these

Such a mechanism

When the highly

that

residue

structures

binding

was ascribed

we suggested

glycopeptides

uniformly

binding

present.

monosaccharides

excess

oligomers

binding

AND BIOPHYSICAL

The enhanced

binding,

regard,

complex

complex

s:te.

one saccharide

probability

types

BIOCHEMICAL

performed effects

concentration

the Mn-Ca-Con protein Since this

on the

that

was reduced

A concentration

solution. the ~1:2 suggests

In each ratio that

120

might from

glycopeptide-Mn-Caalter

of glycopeptide perhaps

observed

1.7 mM to 1.1 mM,to

was maintained instance,

the

at 0.46

the observed to protein

two molecules

mM

relaxation solution

of Con A can

0.55

Vol. 90, No. 1, 1979

interact

with

was initially more

a single

glycopeptide

observed

in

interesting

further precipitate

to ~0.14

formed

precipitates

peptide

presence

solution

this

is

Con A.

evidently

This

(8)

and Schachter's

chain

glycopeptides

since

such molecules

posited

(3)

for

case for

of branched

of cells

branched

chain

binding

on cell

glycoprotein

receptors

membranes

since

observed

multiple

resulting

These

type

may play small

groups

that

we have as is

that

one function

the capacity

of molecules that

may be a

residues,

suggest

to

may be involved takes

place

such as Con A.

an-important

crosslinked

A,

Con A molecules

binding

proteins

the

on branched

on binding

also

and

of Kobata's

by both

and "capping"

in

residues

may be to provide

whole

to bind

to Sepharose-Con

effect

The results

From the

other

two mannose

individual

of multivalent type

(7).

the observations

well

of a glyco-

role

populations

on the In addition,

in signal of complexed

may be involved.

In summary,

the NMKD results

from

with

ovalbumin

multiple

of "patching'.'

of this

each

can be formed

enhancement

proteins.

in the presence

transduction

molecules

as a

capacity

containing

from

to bind

of ThUS.

Con A required

to two lattice-attached

glycopeptide.

glycopeptides

molecules

to bind

containing

known phenomenon

surface

in order

a large Addition

a dimer

chains

at least

a statistical

the ovalbumin

exogeneous

the well

that

affinity

plus

molecules

chain

crosslink

is

(9)

A:

work.

the protein

explain

was

glycopeptide to

At pH 5.6

may be able

effect

ovalbumin

of the

a lattice

An even

the precipitate.

observation

enough

stirring.

standing.

the first

may well

The highest

upon

in future

far

cloudiness

of glycopeptide

of glycopeptide

investigated

must be present

simultaneously. of this

ratio

Thus,

group

upon

in dissolving

of Con A, the

spatially

observation

a small

mM Mn-Ca-Con

was stable

the two branched

two Con A molecules.

group

result

excess

be carefully

knowledge,

are

which

of 0.46

resulted

The precise

will

precipitate.

peptide

mM in the

of a sufficient

residues

crosslink

in

concentration

of the glycopeptide,

mannose

the

as the

quickly

to precipitate

structure

solution

RESEARCH COMMUNICATIONS

Furthermore,

occurred

the protein.

To our

molecule.

disappeared

in the

precipitation

AND BIOPHYSICAL

which

a-D-mannopyranoside

in the presence

for

the

phenomenon

reduced

methyl

BIOCHEMICAL

for

Mn-Ca-Con

the

interaction

A are

121

similar

of a branced to those

found

chain for

glycosimple

Vol. 90, No. 1, 1979

BIOCHEMICAL

saccharides

binding

the changes

in the NMRD spectrum

of Con A by the structure

glycopeptide

of branched

Acknowledgments.

Watson

and use of their Atkinson

supported Welfare

chain

I would

at the IBM T.J.

Paul

to the protein,

for

his

gift

by the National (CA-16054).

ment Award

but are

Curtis

(l-KO4-CA-00184)

are

not

different

a clue

RESEARCH COMMUNICATIONS

in that

as great.

may provide

the magnitude

Secondly, to the

of

the precipitation

functional

aspects

of the

glycopeptides.

like

to acknowledge

Laboratories,

facilities

AND BIOPHYSICAL

for

Yorktown the

S.H.

Heights,

NMRD measurements.

of the purified Cancer

Drs.

ovalbunrin

Institute,

F. Brewer

is

Department a recipient

Koenig N.Y.,

and R.D. for

I would

their like

glycopeptide.

Brown,

III

support to thank This

work

Dr. was

of Health,

Education

and

of a Research

Career

Develop-

from DHEW.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Lis, H. and Sharon, N. (1973) Annual Revs. Biochem. 42, 541-574. Goldstein, I.J., Hollerman, C.E. and Smith, E.E. (1965) Biochemistry 4, 876-883. Brewer, C.F. and Brown, R.D.111 (1979) Biochemistry 18, 2555-2562. Brown, R.D. III, Brewer, C.F. and Koenig, S.H. (1977) Biochemistry 16, 3883-3896. Yariv, J., Kalb, A.J. and Levitzki, A. (1968) Biochim. Biophys. Acta 165, 303-305. Koenig, S.H., Brown, R.D. III and Brewer, C.F. (1973) Proc. Nat. Acad. Sci. 70, 475-479. McKenzie, G.H., Sawyer, W.H. and Nichol, L.W. (1972) Biochim. Biophys. Acta 263, 283-293. Ogata, S., Muramatsu, T. and Kobata, A. (1975) J. Biochem. 78, 678-696. Narasimhan, S., Wilson, J., Martin, E. and Schachter,H. (1979) Can. J. Biochem. 57, 83-96.

122