Isolation and initial characterization of human basement membrane collagens

Vol. 76, No. 2, 1977

ISOLATION

BIOCHEMICAL

AND INITIAL

CHARACTERIZATION

Robert Experimental Pathology Institute/Massachusetts Massachusetts 02114

Received

April

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

OF HUMAN BASEMENT MEMBRANE COLLAGENS

L. Trelstad

and Karen

R. Lawley

Laboratory, Department of Pathology, General Hospital, Harvard Medical

Shriners Burns School, Boston,

13,1977

SUMMARY: Neutral solutions of pepsin extracted human collagens derived from glomeruli, kidney, aorta, lung, heart, bowel, spleen, skeletal muscle and skin were subjected to heat gelation at 370C. Centrifugation of the gel provided two fractions: gelled pellet and non-gelled supernatant. Analysis of these two fractions by gel electrophoresis, molecular sieve and ion exchange chromatography, and amino acid and carbohydrate determinations indicated that the non-gelled supernatant contained a substantial enrichment of basement membrane like collagen. The initial characterization of lung derived basement membrane collagen indicated close similarities with those derived from glomeruli and whole kidney and differences with that obtained from the spleen. INTRODUCTION: which

lie

Basement

at the

membrane

of the

glycoproteins

interface cell

component

the prototype

a unique

collagen

collagen

types

and relative

inaccessibility

study

composition

of its

proteolytic

solubilized

basement

unusual

solubility

found

in the We report

Copyright All rights

interstitium

difficult.

reports

here

pepsin

It

lens

collagens,

composition

material have made use of

by fractionation

non-renal

of the

has expedited

their

and non-ocular species

is

to be

of the

membrane

precipitation

that

capsules

was known

of starting

in basement

a new molecular

whose

the successful

plasma

materials,

and from

Paucity

followed

salt

contain

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

pure

indicate

and glomeruli

like

of the heterogeneity

For interstitial

with

properties,

collagen

collagens.

(4).

of tissues

membranes

lens

membrane

by differential

Recent

(4,5,6).

materials

and the external

glomeruli

the discovery

digestion

collagens

bearing

the

include

surface

(1,2,3).

of basement

in

of cell

matrix

from purified

even before found

a class

composition

and glycosaminoglycans

represents

study

represent

of the extracellular

and whose

The collagenous

limited

membranes

tissues

of collagen

different

from

with

those

(1,7,8,16). isolation

of human basement

membrane

collagens

376 ISSN

0006-291

X

Vol. 76, No. 2, 1977

from a number spleen

of tissues

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

characterization

partial

of those

from

lung,

and kidney.

Human tissues were obtained at autopsy within 12 MATERIALS AND METHODS: hours post mortem. Glomeruli were prepared from kidney slices and isolated from tubules by a graded sieving technique (9). The tissues studied were kidney and glomeruli, liver, lungs, spleen, bowel, skeletal muscle, cardiac muscle, aorta, skin, brain and whole blood. Tissues were homogenized in 0.5M acetic acid adjusted to pH 2.0 to 2.5 with HCl and digested with pepsin (Worthington 2X crystallized) at 20-40 mg/gm wet weight for 3 days at 4oC. The pepsin digest was centrifuged and the extracted collagen solution neutralized with NaOH to pH 7.5 to inactivate pepsin and the solubilized collagens were then precipitated by the addition of NaCl to a final concentration of 20% w/v. The precipitated collagens were collected by centrifugation and the pellet resuspended in 0.4 ionic strength potassium phosphate buffer pH 7.6 and dialyzed against the same buffer. The neutral collagen solution was then placed in a 37O incubator for 4-16 hours to The heat gel was centrifuged at room temperature at effect heat gelation. 6,000 RPM for 30 minutes and the supernatant separated from the pelleted gel. The gel pellet was resolubilized in O.lM acetic acid and resalted out by addition of NaCl to a final concentration of 10% w/v. The collagens in the heat gel supernatant were precipitated by the addition of NaCl to 20-30% w/v. The precipitate was collected following centrifugation and resolublized in either 0.4 ionic strength phosphate buffer or O.lM acetic acid. The portion in phosphate buffer was reduced at 4oC for 16 hours by addition of mercaptoThe reduced collagens were then ethanol at a concentration of 0.1% v/v. to pH 2.0-2.5 with HCl, which dialyzed against 0.5M acetic acid, adjusted When equilibrated at acid pH, also contained 0.1% mercaptoethanol v/v. pepsin (20 mg/ml solution) was added and the collagens were digested at 4OC Following repepsinization the solution was neutralized and the for 16 hours. collagens salted out with NaCl at 20% w/v. For storage of both the heat gel precipitates and supernatants the solutions were dialyzed against O.lM acetic acid and stored in solution. The collagens were analyzed for amino acid and carbohydrate composition, capacity to form SLS (segment-long-spacing) crystallites, electrophoretic mobility on acid-urea and SDS polyacrylamide gels and chromatographic behavior on molecular sieve columns (6% Agarose equilibrated with 5.OM or 8% Agarose guanidine-HCl, 0.05M Tris-HCl pH 7.5, O.OOlM dithiothreitol cation equilibrated with l.OM CaC12, 0.05M Tris HCl pH 7.5) and on CM-cellulose exchange columns all as previously described (10-15). RESULTS: readily

The effectiveness assayed

precipitates. the ratio

by amino Differences

of hydroxylysine

of heat acid

analysis

between

gel

ratio

hydroxyproline/4-hydroxyproline Conversely found

in

the

heat

interstitial

gel

of the heat

the

In addition,

supernatant.

as a fractionation

the heat

precipitate collagen

is gel

of greater shows values

Types

I,

II

377

method

supernatants

and III.

is

and

can be seen by comparing

as shown in Table

generally

ratio

gel

two fractions

to I-hydroxyproline

hydroxylysine/4-hydroxyproline heat

gelation

greater

than

supernatant than

0.04

The

I. 0.35

for

the

generally

has a 3-

and as high

as 0.10.

more consistent

with

those

Vol. 76, No. 2, 1977

BtOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Ratio

I

of hydroxylysine/4-hydroxyproline after

heat

in native

gelation

collagens

fractionation

Kidney

Spleen

Aorta --

Bowel

Lung

Heart ---

Skin

Liver

Supernatant

0.40

0.39

0.39

0.35

0.38

0.38

0.25

0.33

Precipitate

0.14

0.15

0.06

0.07

0.11

0.13

0.08

0.08

Figure 1: Acid-urea polyacrylamide electrophoretogram of collagens from kidney (K), spleen (S), bowel (B), aorta (A), heat gel supernatant, t heat gel precipitate. 1 indicates material from the Agarose column (Figure 3). The gels were times, but those from the same tissues simultaneously. The o(l (I-III) is indicated by a closed square.

Electrophoretic a clear

difference

components reduced gel

top,

Staining

mobility between

are present

and repepsinized and components the

gels

with

in 7.5% polyacrylamide the

in the

two heat acid-urea

in solution: migrating periodic

gels

gel

fractions

gels

of materials

a major between

acid-Schiff

378

standard revealed

at acid

(Figure

component Rana

heat gelled and lung (L). X is the peak 1 run at separate position of

pH indicates

1).

Several

which

had not been

remaining

at the

ti chains

(Figure

prominent

staining

1). of

BIOCHEMICAL

Vol. 76, No. 2, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a Figure 2: SDS-polyacrylamide electrophoretogram of heat gelled kidney a) Heat collagens showing effects of native reduction - repepsinisation. gel supernatant b) Heat gel supernatant after native reduction - repepsinization c) Heat gel precipitate. All samples were reduced after denaturation. The position of o(l (I-III) is indicated by a closed square.

all

bands,

chains.

particularly Electrophoresis

repepsinisation could

greater

ular

prominent

and lesser behavior

weights

migrating

of the material

on SDS gels although

the gels.

Following

components than

were

standard

of collagens

of these

materials

unsuccessful

be seen in

several

anomalous

faster

was usually

on occasion

however, both

the

and/or

materials

will

without

faint

2).

glycoproteins further

the

reduction

d

and

of materials

and repepsinisation,

in 5.0% gels

(Figure

require

above

amounts

reduction

present

chains

present

Owing

with

mobilities

to the

in SDS gels,

the molec-

confirmation

by other

methods. Molecular

sieve

obtained

from

revealed

at least

chromatography

the kidney four

in 5M guanidine

HCl,

at the expected

elution

and not

on Agarose subjected

material

position

eluted

(Bio-Gel

at the void

of a gamma chain

379

gel

supernatant

collagens

to reduction-repepsinisation

On 6% Agarose

components. pH 7.5,

of heat

followed

A-5M), volume

equilibrated of the

by material

column, at

BIOCHEMICAL

Vol. 76, No. 2, 1977

3

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

VO

j.0

8

@

Q

I11

1 0.2

1

I 0.4

1 0.3

I 05

1 0.6

Figure 3: Chromatogram of the heat gel supernatant Agarose A5M equilibrated with 5.OM guanidine - Xl, position of standard )5 , 8 ando( chains are noted. of each of these peaks is given in Table II.

positions This

of an o( chain

pattern

pH 8 with

was not reducing

chromatographic

urea

agents

with

to precipitate

even

gels

suggesting conditions.

column

daltons)

by prior with

were

or without obtained

after

heating

eluting (Figure

a pattern

3, peaks similar

the presence

slightly

before

distinct

band between

to 100°C for volume

to that

standard

eluting o( chains

the

standard

(Bio-Gel solvent

/3

and

380

Similar

the material

The large from

under

3, peak Oc chains

the

6%

in acid1)

the chromatographic

column

in the region

3) electrophoresed (not

molecular

(Figure

material,

the Agarose

(Figure

at

on electrophoresis

of unfractionated

from

protein

A-1.5M)

and the #fraction

aggregates

3).

caused

15 minutes.

1 and 2) revealed

of persistent

The material

this

(Figure

repepsinization.

on 8% Agarose often

peptide

of the denatured

subsequent

but

in the void

of kidney collagen on pH 7.5. The elution The amino acid composition

and a smaller

treatment

1M CaC12, pH 7.5,

materials

Agarose

altered

profiles

equilibrated

weight

(100,000

1 0.0

I 07

shown).

as a The fourth

BIOCHEMICAL

Vol. 76, No. 2, 1977

Amino Acid

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table II Composition

in Residues/1000

NATIVE Kidney -Gel

3-OHPRO I-OHPRO ASP THR SER GLU PRO GLY ALA l/2 CYS VAL MET ILEU LEU TYR PHS OHLYS LYS HIS ARG

peak

:Non-gel 5

from

methylcellulose

column

demonstrated the

acetate,

pH 4.8)

followed

sodium

acetate

gram.

Reduction

graphic

pattern. Amino

acid

with

kidney

given

spleen

show clearcut

lesser

heat

strength

gel

gel

eluting

amounts

of the

in Table

precipitate.

acid-urea

II.

not

native These

differences Surprisingly

components

of material

starting

separated

between

buffer

alter

analyses gel

5 33

after

of the

chro-

(0.02M

sodium

the chromatothe

supernatant were

on the Agarose

column.

chromato-

from the Agarose

from kidney,

few differences

381

in

and fractions

the heat

1

of 0.05-0.06M

later

substantially

selected

6 123 57 26 46 92 70 329 31 5 22 11 22 48 8 18 47

on carboxy-

concentration

materials

8 119 52 22 48 85 62 328 32 3 27 15 28 51 8 20 49 9 6 28

3) - 4

system.

supernatants

present

(Fig. 3

detected

at the beginning

of the

did

Peaks - 2 12 120 52 27 32 94 68 345 31 4 26 13 18 48 8 21 43 1 6 31

successfully

by a peak at a buffer

analyses

are

3) was not

material ionic

11 129 52 22 32 96 68 331 35 2 19 13 20 50 9 24 48 2 6 31

32 12 23 54 21 28 27 17 10 26

in the

and pepsinization

column

four

low

Aqarose

-i

11

(Figure

of denatured

despite

among the

7 69 55 31 43 83 54 356 41

54 25 36 85 62 326 41 8 29 13 24 55 13 27 39 16 10 31

matogram

gel

2 107 55 26 41 90 84 302 38 5 27 14 24 49 15 25 41 15 7 33

and electrophoresis

Chromatography

heat

Spleen Non-gel

97

the Agarose

concentration

Lung Non-gel 9

113 50 18 38 71 90 305 98 2 28 11 17 32 1 15 16 27 8 55

DENATURED

found

lung

and

and the in composition

The values

BIOCHEMICAL

Vol. 76, No. 2, 1977

obtained

from

essentially

the heat

gel

identical

AND BIOPHYSICAL

supernatant

to those

fraction

for

the heat

RESEARCH COMMUNICATIONS

of the isolated

gel

supernatant

glomeruli

were

from the whole

kidney. Amino natants

acid

analysis

(2N NaOH for

lysine

residues

material

for

analyzer

demonstrated

1)

its

digestion

after

to precipitate for

native

The heat the

sample

formed

different

(18,19).

has not

in respect been

structures

requires

comparison

from

the placenta

Chung et al.

is

those quite

(8) although reported possible,

strengths,

capacity not

I,

these

workers that

currently

which

then

in use in the including

has been

actin

and

studied (20),

but

membrane

rich

method. of basement

as a basement materials organs

basement

the basement

membrane

(1,16).

and the

collagen

The recently

by Burgeson

membrane

(1,16,17)

382

unless

fibrillogenesis

are prototypes

possibly

SLS crystallites.

and III.

of collagen

purification

its

conditions

macromolecules

of collagen

2)

and those II

by:

and

SLS crystallites

method

reaction

and internal

however,

form

form

of Types

of material

by other

to

and repepsinization

problem

with

acid

to peptic

ionic

did

tissue

susceptibility

low and high

and glomeruli

and identification

collagens

It

capsule

the amino

in the

denaturation

gelation

to the

same

was indicated

heat

polymerizing

used as a principal

The lens

present

The heat

initially

a purification

different

of

10 minutes)

crystallites is

super-

90% of the hydroxy-

collagens

and its

collagens

Heat gelation

extensively

both

to reduction from

gel

of the

column

supernatant

and 3) its

supernatant

of several

tubulin

from

collagen

gel

DISCUSSION: isolation

at both

was subjected

were

gel

and repepsinization for

over

of hydrolysates

on a short

digestion,

(50°c

that

heat

of glucosamine.

of the heat

gentle

of kidney

indicated

Analyses

the presence

to peptic

reduction

typical

at 110')

in 6N HCl at llO"

state

following

hydrolysates

glycosylated.

4 hours

resistance

capacity

24 hours

were

The native

of alkaline

et al.

in origin data

membrane

at isolated

(7) and

are different presented

collagens

here. from

Vol. 76, No. 2, 1977

different

tissues

has now been

represent

found

for

The basement of similar graphy nents

in the

chains

andfi

Agarose but

prior

heat

than

(7),

the

collagens

(1,16,17).

This

to isolation, studies

questions

size

supernatant

which

resemble

the pattern

which

materials

however,

diversity

in the molecules

do not

with

chromato-

two major

remained

reported lower

at the gel

fractions

compo-

o(l(I-III)

apparent

from

moletop.

the

the A and B ohains,

composition

tissues

of molecules

of A and B chains

proteolysis

and purification

at each of these

to what

Agarose are

from glomeruli

limited

extraction

a group

between

resemble

described

and/or

there

and the

of collagen

are

mobilities

and a fraction

previously

directed

SDS gels

materials

here,

and during

here

as shown by both

with

similar

(4).

moving

starting

of molecules

reported

On the

two faster

of the kidney

differences

collagens

gel

o( chains

reported

synthetic these

et al.

like

class collagens

electrophoresis.

columns

rather

tumor true

kidney

weights

interstitial

but dissimilar

components

by Burgeson

composition

the

composition,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a heterogeneous

membrane

and SDS gel

cular

BIOCHEMICAL

should

and the murine and size

may reflect

both

in situ

(2,8,21). help

Bio-

clarify

(22,231.

Acknowledgements: Reduction-repepsinization was suggested by discussions with Drs. Dehm, Clark, Timpl and Martin at a Symposium on the Biology and Chemistry of Basement Membranes, Philadelphia, 1976 organized by Dr. Kefalides. Supported by the Shriners Burns Institute and Grants from the NIH (HL 18714 and AM 18729). References Kefalides, N.A. (1975), J. Invest. Dermatol. 65:85-92. Hudson, B.G., and Spiro, R.G. (1972), J. Biol. Chem. 247:4229-4238. Trelstad, R.L., Hayashi, K., and Toole, B-P. (1974). J. Cell. Biol. 62:815-830. E.J. and Matukas, V.J. (1974). Fed. Proc. 33:1197-1204. 4. Miller, 5. Trelstad, R.L., Catanese, V.M. and Rubin, D.F. (19761, Analyt. Biochem. 71:114-118. 6. Epstein, E.H. (1974), J. Biol. Chem. 249:3225-3231. 7. Burgeson, R.E., El Adli, F.A., Kaitila, 1.1. and Hollister, D.W. (1976), Proc. Natl. Acad. Sci. USA 73:2579-2583. 8. Chung, E., Rhodes, R.K. and Miller, E.J. (1976), Biochem. Biophys. Res. Commun. 71:1167-1174. 9. Misra, R.P. (19721, Amer. J. Clin. Path. 58:135-139. 10. Trelstad, R.L. and Lawley, K.R. (19761, Analyt. Biochem. 70:287-289. 11. Ortec Application Note AN 32A (1973) p. 39. 12. Stark, M. and Kuhn, K. (1968), European J. Biochem. 6:534-541.

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Vol. 76, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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