Iozzo Renato V. Proteoglycan Protocols

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Amyloid Peptide: GAG Reactions 449

449

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Serum Amyloid A Peptide Interactions

with Glycosaminoglycans

Evaluation by Affinity Chromatography

John B. Ancsin and Robert Kisilevsky

1. Introduction

Amyloid is a pathological deposit of protein and glycosaminoglycans (GAGs) that

can lead to the destruction of tissue architecture and function (1,2). To date, 18 unre-

lated proteins are known precursors of amyloid deposits (3). Each one is associated

with a specific disease, such as Alzheimer’s disease, adult–onset diabetes, inflamma-

tory disorders, and some cancers, but regardless of the type of precursor protein

involved, all amyloids have common tinctorial and structural characteristics. Fibrils

are composed of two or more filaments 3 nm in diameter twisted around each other

forming nonbranching fibrils 7–10 nm in diameter with a crossed β–pleated sheet con-

formation. Congo red stains these deposits, and when viewed under polarizing light

they exhibit a red/green birefringence characteristic™ for amyloid.

Available evidence suggests that the deposition of amyloid involves a nidus or

protofilament around which amyloid fibrillogenesis occurs, and heparan sulfate (HS)

facilitates this pathological process (2,4,5). To understand better the mechanism by

which this HS–dependent transformation takes place, a number of studies have been

undertaken to characterize potential amyloid precursor–HS–binding activities. Hep-

arin–HS–binding activity has been reported for five amyloid precursors, Aβ and βPP

(6,7), tau (8,9), prions (10,11), amylin (12,13) and serum amyloid A (14). The latter is

an acute–phase apoprotein of HDL for which we have recently defined its GAG–bind-

ing site.

Heparin/heparan sulfate–binding activity for serum amyloid A (apoSAA) was

localized to its carboxyl–terminal end, residues 77–103, by affinity chromatography

450 Ancsin and Kisilevsky

of apoSAA2 1.1 CNBr fragments. The relative importance of the six basic residues

within this region was demonstrated by employing different mutant synthetic peptides

in which one or more of these basic residues were either deleted or replaced with

alanine. The GAG–binding potential of protein/peptides is generally tested only on

heparin, but we were interested to see if apoSAA had an affinity for any other GAGs.

Chondroitin sulfate, dermatan sulfate, hyaluronan, and heparan sulfate charged col-

umns were generated with Sepharose 4B and Affi–Gel and their binding affinity

tested. In addition, the influence on binding activity of two popular coupling reactions

used to link GAGs to column matrices were compared and found to have an influence

on apoSAA binding.

2. Materials

2.1. Glycosaminoglycan Charged Columns

1. Heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, and hyaluronan were all

purchased from Sigma (St. Louis, MO). Sepharose 4B was purchased from Pharmacia

Biotech (Baie d’Urfé, Quebec), 1–ethyl–3–3–dimethyl–aminopropyl carbodiimide

(EDAC), Heparin–Affigel and Affigel–102 were purchased from Bio–Rad (Hercules, CA).

2. Glycosaminoglycan assay reagents: 0.2% NaCl; GAG standards (1 mg/mL in 0.2%

NaCl); 0.005% toluidine blue (5 mg/100 mL 0.01 N HCl, 0.2% NaCl); ethanol (abso-

lute) and hexane.

3. Other reagents: 50 mM acetate pH 6.0, 20 mM Tris–HCl, pH 7.5, 1 M ethanolamine, pH

9.0,0.1 M sodium acetate, pH 4.0, 0.1 M NaHCO

, pH 8.3, cyanogen bromide (Sigma)

stock solution 1 g/mL in N, N–dimethylformamide, 70% formic acid, and N

2 (gas).

4. Waters HPLC system (Waters Chromatography, Milford, MA) with a model 680 auto-

mated gradient controller, model 501 pump units, series 440 absorbance detector connected

to a Waters 740 data module integrator.

3. Methods

3.1. GAG–Sepharose Columns

Cyanogen bromide (CNBr) activation is a common method used in the preparation

of affinity resins. It is a relatively simple reaction that works well with agarose–based

column matrices and has minimal secondary effects on ligand chemistry. Briefly, at

alkaline pH the hydroxyl groups on the agarose resin react with CNBr. A high molar

excess of CNBr is required, because most of it (1) reacts with water, generating inert

cyanate ions, or (2) forms cyanate esters, which become hydrolyzed, or (3) reacts with

matrix hydroxyls to form imidocarbonates. The remaining active cyanate esters react

with amino groups on lysines of proteins, or hydroxyls on carbohydrate chains.

1. GAGs were linked to Sepharose 4B based on the method of Smith et al., (15). Sepharose

4B was washed with 20 bed volumes of water, resuspended in 1 volume of water, and

transferred to a beaker with a stir bar on ice. GAGs were dissolved in water at 2 mg/mL

and also placed on ice.

Amyloid Peptide: GAG Reactions 451

2. The two solutions were mixed and, while stirring, the pH was adjusted to pH 11 with

NaOH (5 N). Cyanogen bromide, 1 g/mL in N,N–dimethylformamide, was added

dropwise to a final concentration of 31.3 mg/mL.

3. The pH was maintained at about 11 by adding NaOH for 15 min, then left stirring for 18

h at room temperature. The pH gradually returns to approximately pH 8.

4. The gel was then washed with 20 bed volumes of water followed by 1 M ethanolamine,

pH 9.0, to block excess reactive groups. After further washing with 10 bed volumes of (1)

water, (2) 0.1 M sodium acetate pH 4.0, and (3) 0.1 M NaHCO

, pH 8.3, the column gel

was equilibrated in 20 mM Tris–HCl, pH 7.5.

3.2. GAG–Affigel Columns

GAGs can be covalently linked to agarose matrices by activation of the uronic acid

carboxyls with EDAC coupling the GAG chain to an amino–functionalized matrix

such as Affi–Gel–102 (Bio–Rad) (see Notes 3 and 4).

1.Affigel–102 (4 mL) was washed with 20 bed volumes of 50 mM acetate, pH 6.0, and then 8

mg of GAG in 4 mL of the same buffer was mixed with the gel.

2. The coupling reaction was initiated by the addition of 32 mg of EDAC, adjusting the pH to

5 with 1 N HCl and allowing the reaction to proceed for 3 h at room temperature.

3.3. Assay of GAG Coupling Efficiency to Matrix

(Toluidine Blue Method)

1. Add 0–35 µL of GAG to tubes containing 0.75 mL of 0.2% NaCl—this is your standard.

Also dilute the GAG column matrix 1/4 with 0.2% NaCl (0.75 mL total). Blank tubes

should contain 0.2% NaCl or Sepharose 4B in 0.2% NaCl.

2. To all tubes, add 0.75 mL of toluidine blue solution and vortex for 30 s. Next add 1 mL of

hexane to each tube and vortex for another 30 s. GAG–dye complexes should precipitate at

the water–hexane interface. Centrifuge at 735g (3000 rpm) for 5 min. Aspirate the hexane

and the top half of the water layers and discard. Dilute a sample of the water layer 1/10 with

absolute ethanol, mix, and read at 631 nm within 30 min. Care must be taken not to get any

of the GAG–dye complex precipitate into the sample to be assayed.

3. The amount of GAG linked to columns ranged from 0.5 – to 0.75–mg/mL of gel.

3.4. Affinity Chromatography on GAG–columns

3.4.1. Low–Pressure Liquid Chromatography

1. Peptides were dissolved in 20 mM Tris–HCl, pH 7.5, and loaded onto a 6–mL Heparin

Affigel column preequilibrated in the same buffer (see Fig. 1).

2. The column was then washed at 30 mL/h with 4 bed volumes of buffer followed by a

0–1 M NaCl linear concentration gradient. Fractions were collected (0.6 mL) and their

absorbency determined at 214 nm (see Notes 5 and 6).

3.4.2. High–Pressure Liquid Chromatography (HPLC)

1. Different GAG–charged matrices were also packed into a 3–mL stainless steel column

and equilibrated with 20 mM Tris–HCl, pH 7.5, at 0.5 mL/min using a Waters HPLC

system (see Figs. 2 and 3).

452 Ancsin and Kisilevsky

2. Samples (20–80 µg in 150–200 µL) were injected onto the column, washed with 3 bed

volumes (18 min) of the same buffer, and then developed with a 0–1.0 M NaCl linear

gradient for 10 bed volumes (60 min). The eluate was monitored continuously at 214 nm

and the absorbance plotted against the retention time (RT).

3. Generally, unbound peptides/proteins eluted 6.5–7.0 min after loading and, based on the

RTs for the bound peptides/proteins, the NaCl concentration at which desorption took place

was calculated; desorption [NaCl] = (RT –6.5 min –18 min) / 60 min.

3.5. ApoSAA2 Peptides

3.5.1. CNBr Peptides

1. ApoSAA1.1 was cleaved at Met–X peptide bonds with cyanogen bromide (CNBr).

Protein was dissolved in 70% formic acid at 1 mg/mL, to which 5.5 mg/mL CNBr

was added (250 molar excess over Met) plus free

L–Trp (5 molar excess over Met) to

protect Trp residues.

2. The reaction was carried out under nitrogen, at room temperature overnight, and then the

solvent was evaporated by vacuum centrifugation.

3. The reaction was evaluated and peptides purified, by reverse–phase (RP)–HPLC. A

semipreparative C–18 Vydac column was equilibrated with 10% acetonitrile, 0.1%

Fig. 1. Elution profile of the apoSAA1.1 CNBr peptides on a heparin–Affigel column: testing

apoSAA1.1 CNBr fragments for heparin–binding activity. ApoSAA1.1 CNBr cleavage fragments

are shown above the graph; 16mer (residues 1–16), 7mer (residues 17–23), 53mer (residues

24–76), and 27mer (residues 77–103). A heparin–binding consensus sequence, (XBBXBX,

X = nonbasic, B = basic) is underlined in m27mer.

Amyloid Peptide: GAG Reactions 453

TFA. Peptides were dissolved in 40% formic acid, filtered through a 0.2–µm filter, or

centrifuged at 10,000g, and loaded onto the column, washed for 5 min at 3 mL/min,

then developed with a 1.5%/min acetonitrile linear concentration gradient for 30 min,

followed by 4.3%/min acetonitrile for 10 min, bringing the elution buffer to 98%

acetonitrile by 45 min.

4. The separated peptides were identified by amino–terminal sequencing and molecular–

weight determination by mass spectroscopy carried out at the Alberta Peptide Institute

(Edmonton, Alberta, Canada).

3.5.2. Synthetic Peptides

1. A series of eight apoSAA1.1 peptides corresponding to wild–type mouse apoSAA1.1,

residues 77–103, (m27mer) six peptides in which one or more of the basic residues were

replaced with A, and one 20mer residues 77–96 (missing K102) were synthesized by

Multiple Peptide Systems (San Diego, California, U.S.A.). (see Notes 1 and 2).

Fig. 2. Identification of the basic residues of m27mer that are important for heparin binding.

Chromatography was performed on heparin–Sepharose 4B and the elution profiles were simi-

lar to that seen with heparin–Affigel (not shown) with the exception of K102–. Carbodiimide

treatment of HS interferred with binding (HS–Sepharose + carbodiimide). The sequence of

m27mer is shown above the graph.

454 Ancsin and Kisilevsky

m27mer (mouse apoSAA1.1

77–103

) ADQEANRHGRSGKDPNYYRPPGLPAKY

R83A ADQEAN

AHGRSGKDPNYYRPPGLPAKY

H84A ADQEANR

AGRSGKDPNYYRPPGLPAKY

R86A ADQEANRHGASGKDPNYYRPPGLPAKY

R83A,H84A,R86A ADQEAN

AAGASGKDPNYYRPPGLPAKY

K89A ADQEANRHGRSG

ADPNYYRPPGLPAKY

R95A ADQEANRHGRSGKDPNYY

APPGLPAKY

K102– (mouse apoSAA1.1

77–96

) ADQEANRHGRSGKDPNYYRP

2. Purity of the peptides was analyzed by RP–HPLC and, where required, the peptide was

re–purified.

4. Notes

1. To localize GAG–binding sites to specific peptide sequences, limited chemical or enzy-

matic cleavage is required of the native protein. Additional flanking sequences to main-

tain appropriate secondary structure and cleavage near basic residues (K, H, R) should be

avoided. Chemical cleavage at M–X with CNBr, or at W–X with BNPA–skatole is a good

starting point, since both residues occur with relatively low frequency in proteins (M 2.4%

and W 1.3%) (16).

Fig. 3. Determination of the GAG–binding specificity for m27mer. Heparan sulfate (HS),

chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronan (HA) were covalently linked to

either Sepharose 4B of Affigel 102. The elution profiles on DS–Sepharose and HA–Sepharose

(asterisks) were very similar to that of CS–Sepharose.

Amyloid Peptide: GAG Reactions 455

2. Whenever possible, conservative residue replacements should be performed. The strat-

egy for deciding on replacement of residues in GAG–binding sites should take into

account potential changes in peptide solubility, secondary structure (i.e., conformational

preferences α–helix, β–strand, β–bend turn), and the volume of the amino acid chosen.

The basic residues most often shown to be crucial for GAG binding (K, H, R) are α–helix

formers (16), and A can be a satisfactory replacement. However, if solubility of the pep-

tide is adversely affected then Q may be a useful alternative. Also, Qs van der Waals vol-

ume is more similar to K, H, and R than A; Q = 114 Å

vs A = 69 Å

, compared to K = 135

, H = 118 Å

, and R = 148 Å

3. All GAGs, CCS, DS, HA, HS, and heparin can be covalently coupled to column matrices,

and determination of the GAG–binding specificity of any peptide using these columns

can be easily carried out. Heparin is quite popular and inexpensive, and is often used as a

cheap substitute for HS. While heparin and HS are synthesized in a similar fashion,

sulfation and uronic acid epimer (glucuronic vs iduronic acid) content and distribution

are quite different. Also HS is most often the physiological ligand for most proteins and

peptides that can bind heparin. We have observed that while heparin–binding activity

was useful in localizing the GAG–binding site on apoSAA1.1, the minimal peptide

sequence required for binding to heparin was at least 7 residues shorter then that required

for HS binding. In addition, deletion of K102 destroyed binding for HS–Sepharose, but

not for heparin–Sepharose implying that the K102–peptide was binding to different oli-

gosaccharide sequences.

4. Type of support matrix. Sepharose vs Affigel helps define the type of chemical linkage

group available to react with one or more of the GAG’s reactive side groups. This is an

important factor, since the linkage of GAG to matrix can influence binding activity. We

found that the basic residue requirements were found to be different for heparin, depend-

ing on whether it was linked via its COO– or HO– groups. We observed that K102–bound

heparin–Sepharose but not heparin–Affigel. A similar discrepancy was seen with the

m27mer peptide, which bound HS only when linked to Sepharose, not Affigel. Further-

more, treatment of HS–Sepharose with carbodiimide indicated that blocking of the car-

boxyls on HS was likely responsible for the loss of binding activity.

5. Preliminary screening of protein/peptide for GAG–binding activity can be carried out

relatively quickly with 1– to 10–mL columns. Samples can be loaded in 0–0.15 M NaCl,

followed by an increasing linear NaCl concentration gradient to 1 or 2 M NaCl totaling

10–20 bed volumes to develop the elution profile. Distinguishing between different pep-

tides with similar affinities for the GAG column was more readily achieved with the

columns connected to HPLC. The HPLC allowed for more precise solvent delivery and

gradient formation, highly reproducible retention times, and accurate determination of

NaCl concentrations required for protein/peptide desorption.

6. FPLC– and HPLC–grade heparin columns are also available from a number of suppliers.

References

1. Sipe, J. D. (1994) Amyloidosis. Crit. Rev. Clin. Lab. Sci. 31, 325-354.

2. Magnus, J. H. and Stenstad, T. (1997) Proteoglycans and the extracellular matrix in amy-

loidosis. Amyloid: Int. J. Exp. Clin. Invest. 4, 121–134.

3. Kazatchkine, M. D., Husby, G., Araki, S., Benditt, E. P., Benson, M. D., Cohen, A. S.,

Frangione, B., Glenner, G. G., Natvig, J. B., and Westermark, P. (1993) Nomenclature of

amyloid and amyloidosis. Bull. WHO 71, 105–108.

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4. Kisilevsky, R. and Fraser, P. (1996) Proteoglycans and amyloid fibrillogenesis, in The

Nature and Origin of Amyloid Fibrils (Bock, G. R. and Goode, J.A., eds.), Wiley,

Chichester, UK, pp. 58–67.

5. Kisilevsky, R. and Fraser, P. (1997) Aβ amyloidogenesis: unique, or variation on a sys-

temic theme? Crit. Rev. Biochem. Mol. Biol. 32, 361–404.

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and Kisilevsky, R. (1991) High affinity interactions between the Alzheimer’s beta–amyloid

precursor proteins and the basement membrane form of heparan sulfate proteoglycan. J.

Biol. Chem. 266, 12,878–12,883.

7. Leveugle, B., Scanameo, A., Ding, W., and Fillit, H. (1994) Binding of haparan sulfate

glycosaminoglycan to beta–amyloid peptide: inhibition by potentially therapeutic

polysulfated compounds. Neuroreport 5, 1389–1392.

8. Goedert, M., Jakes, R., Spillantini, M. G., Hasegawa, M., Smith, M. J., and Crowther, R.

A. (1996) Assembly of microtubule–associated protein tau into Alzheimer–like filaments

induced by sulphated glycosaminoglycans. Nature 383, 550–552.

9. Hasegawa, M., Crowther, R. A., Jakes, R., and Goedert, M. (1997) Alzheimer–like changes

in microtubule–associated protein tau induced by sulfated glycosaminoglycans—inhibi-

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on degree of sulfation. J. Biol. Chem. 272, 33,118–33,124.

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ecules bind differentially to prion–protein and change their intracellular metabolic fate. J.

Cell Physiol. 157, 319–325.

11. Caughey, B., Brown, K., Raymond, G. J., Katzenstein, G. E., and Threscher, W. (1994)

Binding of the protease sensitive form of PrP (prion protein) to sulfated glycosaminoglycans

and Congo red. J. Virol. 68, 2135–2141.

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amyloidogenic peptides Aβ and amylin. Dependence on aggregation state and inhibition

by Congo red. J. Biol. Chem. 272, 31617–31624.

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K., Hansen, J. B., and Snow, A. D. (1998) Sulfate content and specific glycosaminoglycan

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Lipoprotein Proteoglycan Interactions 457

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From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Interactions of Lipoproteins with Proteoglycans

Kevin Jon Williams

1. Introduction

1.1. General

Significant physiological and pathophysiological processes involve interactions of

specific lipoproteins with specific proteoglycans. So far, these interactions fall into

two large groups. The first is interactions with heparan sulfate proteoglycans (HSPGs),

primarily on the cell surface that lead to cellular uptake of the lipoproteins. These

pathways are of interest because they involve endocytic machinery, intracellular

itineraries, and regulation that are generally distinct from classic, coated pit-mediated

endocytosis (1). Moreover, many important nonlipoprotein ligands, such as infectious

agents, growth factors, platelet secretory products, and proteins implicated in

Alzheimer’s disease, bind to the same HSPGs, and lipoproteins are a convenient model

ligand to study HSPG-mediated catabolism. The second group of lipoprotein-

proteoglycan interactions involves chondroitin sulfate proteoglycans (CSPGs),

primarily in the extracellular matrix, leading to retention of cholesterol-rich lipopro-

teins. Many lines of evidence now support the concept that retention of lipoproteins

within the arterial wall is the key initial step in provoking atherosclerosis, the major

killer in Western countries (2,3t).

The following sections contain a brief primer on general lipoprotein biology and

methods (see Subheading 1.2.); then essential background for methods used to study

lipoprotein-HSPG interactions, with a focus on endocytosis (see Subheading 1.3.);

background relevant to molecular methods to study HSPG-mediated endocytosis, with a

focus on specific domains within the syndecan-1 core protein (see Subheading 1.4.);

and then five detailed, basic protocols (see Subheadings 2–4). Because of space limi-

tations, lipoprotein–CSPG interactions are not covered in detail here, although key

methods can be found in the published literature (4–13).