A collagen of unusual composition and a glycoprotein isolated from canine glomerular basement membrane

Vol. 22, No. 1, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A COLLAGEN OF UNUSUAL COMPOSITION AND A GLYCOPROTEIN ISOLATED FROM CANINE GLOMERUlAR BASEMENT MEMBRANE Nicholas La Rabida-University

of Chicago University

Received

November

in a recent presence

study,

Kefalides

membrane

with

in 8 M urea.

Amino acid

8 M urea

revealed

the presence

basement

membrane

fractions

did

Dlsche

to collagen

from canine

glomerular

a collagen than

from healthy, according

membrane

a collagen-like

protein

in

The basement

0.07

and alkylation membrane

in matmna1ian

hexose, acid

frcm

of Glunerular mongrel

dogs.

with

membrane.

a much higher in other

and its

ratio

of

of

baseacid,

basement

and galactose

the separation

In

of the soluble

in human glomerular

membrane. 1 inked basement

two components

One is a glycoprotein

hydroxyproiine

(0.42

2.1% sialic

intact

of glucose

fractil

Intact

composition

that

bonds

soluble

collagen).

and carbohydrate

deals

basement

is found

was solubilfred of disuifide

1.4% glycosamine,

the presence

report

membrane

hydroxylysine:hydroxyproline

significantly

to the method ion

a)

5.52%

with

Preparation

Extract

the

and of a sialofucohexosaminoglycan The present

content

Jllinois

reported

of basement

with

reported

membrane .

other

of Medicine,

(l864,1865)

or by reduction

The amino

(1965)

with

of dogs.

analysis

canpared

not differ

et al --•

and Winrler

of a high

contained

fucose.

Chicago,

associated

by extraction

and 0.75%

and Department

18, 1965

basement

ment membrane

institute

of Chicago,

of a glycoprotein

glomerular

A. Kefalides

and the

and hydroxylysine

mammal ian co1 lagens. Basement The glanerular

of Krekower

Membrane:

Kidneys

basement

and Greenspon

were

membrane

obtained

was prepared

(1951).

Procedures

Extraction was extracted

with

5% TCA: with

Ten milligrams

4 ml of 5% trichloroacetlc

26

of

lyophilized acid

basement for

1 hour

at

Vol. 22, No. 1, 1966

80’~.

After

residue

BIOCHEMICAL

centrifugation

was washed

washings

added

dialyzed

for

accounted

with

about

membrane

was extracted

Since

Bovine

5% TCA In a similar Extraction

basement

membrane

aqueous

methyl&nine,

water

the original Colunn

with

Bio-Gel

at 37’C 1 hour.

concentration TM ing the

bulk

appeared

for

Total

fractions

Samples

of protein

was thus

chranatowith

amino

acids

Amino

was centrifuged

was dialyzed

against

Approximately

from

buffer,

basement

60% of

pH 8.4,

with

membrane applied

readings

at 280 mp.

The first

fraction

(Fraction

the void

voiune.

One

Protein

density

with

with

to a

the same buffer.

of 3 ml per minute.

A) contain-

The second

52 and 68 ml of eluate.

hexose

hydrolyzed

acid

analysis

analyzer

and Morris

by the orcinol were

0.1%

fraction

Only Fraction

and carbohydrate.

In a Technicon end Piez

to pH 8.5 with

solubilired.

rate

between

of lyophiiized

adjusted

solution

M, Tris-HCl

emerged peak

milligrams

The material

extracted

appeared.

protein-bound

and its

in basement

was extracted

and iyophilized.

by optical

Determinations:

(1854),

24 hours.

at a flaw

broad

hydroiysates

and Stein

collagen

8 M urea,

X 50 cm) and eiuted

of the protein

&hemlcal protein

(1.1

fractions

A was ana’lyted

with

5 days,

in 0.05

collected

as a low,

The residue

by column

One hundred

for

membrane

was measured

major

were

25x of the starting

fractionation tendon

The material

column

were

about

The supernatant

at 4’C for

was dissolved

ml aliquots

residue

of the carbohydrate

Achilles

was extracted

basement

P-300

and the

and the

and iyophiiized.

for

method,

8 M Urea:

Chromatoqraphy:

8 M urea

insoluble

manner.

b)

distilled

water

a majorportion

by this

was attempted.

x g for

distilled

the

centrifuged

the extract

50% and the extract

by weight.

at 34,800

acid,

Both

RESEARCH COMMUNICATIONS

15 minutes,

5% trichloroacetic

5 days against

material

graphy

at 1400 x g for

to the extract.

for

AND BIOPHYSICAL

was carried

employing

out

the methods

on of Hoore

(1960).

was determined reaction

in intact

of Welmer

in 6 E HCl for

27

basement

and Moshin 4 hours

at

membrane

(1953). 1OO’C and the

Vol. 22, No. 1, 1966

amino

sugars

separated

by the method Table

Achilles

tendon,

amino

acids.

It will

basement

hydroxyproline membrane

to Boas (1953).

the anino

of basement

Values

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and Morgan

1 shows

5% TCA.

lysine,

according

of Elson

Results:

with

BIOCHEMICAL

acid

are expressed that

and proline contains

2.2

determined

was

(1933). canposition

membrane

be noted

Hexosamine

and its

in terms

residues

fraction

basement does tendon

frcm bovine

obtatned

of residues

intact than

of collagen

per

membrane collagen.

of hydroxylysine

by extraction

100 residues contains However,

canpared

to

TABLE I AMINO ACID ANALYSIS

OF BASEMENT KMBRANE AND TENDON COLLhGEN*

Achilles Tendon Co1 1agen Aspartic

4.9

Th reon i ne Serine Glutamic

Basement TCA Extract

Membrane Intact

1.7

5.9 2.1

7.3 4.0

3.6 8.0

3.9 8.6

4.9 11 .o

Proi ine

12.5

Glycine

31.2

7.1 31.0

21.0

Alanlne

10.8

5.6

6.7

2.4 0.00

2.7

4.7

1l oo

1.6

Methionine

0.45

0.2

0.9

lsoleucine

1.4

I.8

2.8

Leuc i ne

2.6

4.6

6.7

Ty ros I ne

0.5 1.4

0.5 2.1

3.3

Val ine Cystine

l/2

Phenylalanine

7.0

1.8

Lys I ne

2.4

1.3

2.8

Histidine

0.5

0.6

1.4

Arginfne

5.4

2.9

4.9

HO-Proline

9.9

15.8

5.3

HO-Lys i ne

0.7

4.3

2.2

HO-LYS ine HO-Pro1 ine

0.07

0.27

0.42

*Values

are expressed

in residues

28

per

100 residues.

of less

Vol. 22, No. 1, 1966

0.7

BIOCHEMICAL

in collagen.

tendon

The TCA extract

collagen

glycine

in sane

content

compared

with

proilne

is 31.2

is similar

22.4

in tendon

9.9

(4.3

in tendon

collagen).

residues,

residues The amino

not

differ

that

collagen

up to about

the

Striking

all

hexose

are

tendon

collagen

collagen

membrane. membrane

component

acid

in tendon

is

collagen and

of valine,

with

In contrast,

is similar

to that

aration

of the non-collagen extract,

the

hexose

chromatography

of

8 M urea

in

in carbohydrate

practically

of basement

absent.

and glycine.

was

membrane.

of hexose

content

by chranetography richer

3 shaws the

and the various

portion

membrane.

In

Differences Table

membrane

a large

In a fraction

which

A and the TCA residue.

basement

29

resulted

of the fraction fraction

basement

component results

Colman

and hexosamine

of intact

is contributed

membrane.

lysine

5% TCA removes

I).

makes

A) richer

Fraction

intact

membrane

in Table

the collagen

with

non-collagen

are

included

did

that

composition

between

of

not

(Fraction

from basement

content

(data

can be concluded

basement

noted

fran

in basement

and hydroxylysine

in the content

the 8 M urea

to 0.7

and of the

extract

from

there

compared

it

the amlno

8 M urea

Extraction

hexosam 1 ne .

Similarly,

to

in the TCA extract

of basement

residue

and hexosamine

fractions.

compared

than

tendon

then

from

similarities

are evident

of

in the TCA extract

the hydroxyproline

2 summarizes

hydroxyproline

collagen.

and

residues

in the TCA extract

of the TCA extract

intact

ccmponent,

with

ine and

in the TCA extract

15.8 and 7,l

in tendon

of a non-collagen

Table

both

of

the TCA insoluble

extracted

are

The

collagen).

extracted

isolation

than

there

respectively,

30% by weight

the material

The sum of hydroxyprol

However,

composition

from that

is assumed

by the

acid

in the TCA extract

residues

respectively

resembling

residues

(22.8

lysine

although in others.

proteins

and less

in tendon

membrane,

significantly

(31.0

respectively

and 1.3 residues

2.4

it

in both

in both

more hydroxylysine

differs

identical

and proline,

and 12.5

of basement

respects,

collagen).

hydroxyproline

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

of the 8 II urea Furthermore,

in Bio-Bel

sepP-300

in carbohydrate,

if

Vol. 22, No. 1, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 2 AMINO ACID ANALYSIS OF BASEMENT MEMBRANE FRACTIONS* 8 M Urea Extract 8.00

8.3

8.1

Th reon I ne

4.7

6.0

5.7

Serine Giutamic

6.2 10.6

8.7 13.2

7.3 1 I.4

Proi ine Giycine

6.6 20.0

8.3 13.5

6.8 12.0

6.3

7.6

8.8

4.9

3.8

5.1

1.4

2.9

2.9

Methionine

1.2

0.9

0.8

Vai ine Cystine

i/2

isoleucine

3.8

4.3

3.5

Leuc i ne

7.0

8.9

8.1

Tyrosine

1.9

I .6

2.1

Phenylaianine

3.3

4.2

3.3

Lys i ne

2.8

1 .o

4.0

Histidine

1.9

1.2

1.9

Arginine HO-Proline

5.2 5.0

5.4 Trace

6.0 0.00

HO-Lys ine * Values are

2.0

Trace

Trace

containing

expressed

0.5 wies

hexosamine/mg

suggest

of a collagen,

unique

(1.3%

by weight).

that

obtained

is a giycoprotein coranunications dlffraction

canine

in its

The amounts

the collagen

per

of protein

as much hydroxyproiine

coi lagen.

in residues

hexose/mg

of protein

The data

X-ray

Caponent TCA Residue

Aspartic

Aianine

times

Non-Collagen Fraction A

and six

of proline fran

which (Kefaiides pattern

basement It

times

and Winzier, of basement

are

membrane

one and a half

reduced

wa have

as tendon

by almost

The non-collagen

gives

voles

is made up

and 1.3% hexosamine.

1964,1965)

30

membrane

contains

mambrane.

9% hexose

and 0.074

as much hydroxylysine,

and iysine

basement

contains

(9% by weight)

glaneruiar

canposition.

100 residues.

reported

a “powdarl’

50% in

canponent In previous that diagrim

the with

Vol. 22, No. 1, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 3 CARBOHYDRATE ANALYSIS

OF BASEMENT MEMBRANE

umoles/mg I r&act

8 M Urea Extract

Hexose

0.31

0.32

0.42

0.15

0.50

Hexosamine

0.056

0.06

0.027

0.048

0.074

measurements

at 2.85,

non-oriented

structure.

4.23

is a protein

content

makes up about

proline

plus

then

the fraction

classified

with

content

accounts

for

It

increased

about

2/9

membrane

has been shown

Wood and Keech (1965),

reconstitution

content

(1962)

and whose residues

with

that

glycine

and whose

of the total

hydroxy-

residues,

5% TCA could

that

acid

(1960),

be

fibers.

and polysaccharide

may influence

the

diemeter

average

Although

no collagen

fibers

electron

microscopy,

the data

present

and

is associated

hydroxyproline

elucidate orientation

of basement

Acknowledqements. llaty

with

role

that

indicate

will

affect

the type

and

content. amino

This

might

on

collagen

collagen

Studies acids

--in vivo.

membrane that

is

is unique

are under play

their

wishes

to thank

technical

Mrs.

assistance.

31

(1965)

phase

formation

In basement

here

these

et al.

in the extracellular of fibril

Schmitt

in

way to

in the structural

membrane.

The author

Lou Spach for

that

a glycoprotein.

and hydroxylysine

the possible

is conceivable

have been seen presented

by Grant sulfate

and rate

acids

(1951),

or chondroitin It

of collagen

amino

et al.

and more recently

glycoprotein

of collegen

of hydroxylated

by Highberger

concentration

Miss

acid

helical,

criteria

structure

amino

from basement

of the

clear.

(1955),

and Mathews

its

triple

Ramachandren’s

helical

of the total

with

Fract 1 on-A

as a collagen.

is not as yet

the

a triple

extracted

TCA-Res 1due

8, canpatible

l/3

The significance

et al --•

and lo.27

If one accepts

collagen

proline

TCA-Ext ract

L. Forsell-Knott

and

Vol. 22, No. 1, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES Boas, N. F., J. Biol. Chem., 204:553, 1953. Dische, R. M., Pappas, G. D., Grauer, A., and Dische, i!., Biochem. Res. Camwn., %:63, 1965. E Ison, L. A., and Morgan, W. T. J., Biochem. J., a:1824, 1933. Grant, R. A., Horne, R. W., and Cox, R. W., Nature, =:822, 1965. Highberger, J. H., Gross, J., and Schmitt, F. O., Proc. Nat. Acad.

u:286,

Biophys.

Sci.,

I95 I.

Kefaiides, N. A., and Winrier, R. J., Abstracts of Papers, Am. Chem. Sot, 148th Meeting, p. 4iC, August-September, 1964. Kefaiides, N. A., and Winzier, R. J., 1965. Submitted for pub1 ication. Krakower, C. A., and Greenspon, S. A., A.M.A. Arch. Path., 2:62Y, 1951. Mathews, M. B., Biochem. J., &:710, 1965. Moore, S., and Stein, W. H., J. Bioi. Chem., =:907, 1954. Piet, K. A., and Morris, L., Anal. Biochem., 1:187, 1960. W., and Thathacheri, Y. T., in Colla en Ramachandran, G. N., Sasisekharan, Interscience Publishers, New York, pa -7&k& N. Ramanathan, Editor. Schmitt, F. O., Gross, J., and Highberger, J. H., Symposia 50~. Exp. BiOT., p:i% 1955. Weimer, H. E., and Moshin, J. R., Am. Rev. Tuberc., %:594, 1953. Wood, G. C., and Keech, M. K., Biochem. J., E:588, 1960.

32