Biochemical and Biophysical Research Communications 315 (2004) 297–305

BBRCwww.elsevier.com/locate/ybbrc

A heat shock protein70 fusion protein with a1-antitrypsinin plasma of Type 1 diabetic subjects

Paola Finotti* and Andrea Pagetta

Department of Pharmacology and Anaesthesiology, University of Padua, Largo E. Meneghetti 2, 35131 Padua, Italy

Received 15 January 2004

Abstract

The recent observation that heat shock proteins (HSPs), mostly glucose regulated protein94 (Grp94) and HSP70, are present in

plasma of Type 1 diabetic subjects as complexes with immunoglobulins, prompted us to investigate the nature and extent of this

association, whether it represents HSP-induced activation of the immune system. Two complementary affinity chromatography

procedures followed by immunoprecipitation and immunoblot analyses of HSP-enriched, plasma-purified peaks, revealed that HSPs

were inextricably linked with IgG in SDS-resistant complexes from which proteins dissociate partially under reducing treatment.

HSP70 was found also closely linked with a1-antitrypsin (a1AT) in a single protein having the mass of a1AT but elution charac-

teristics different from those of normal a1AT. Immunoprecipitation with anti-HSP70 antibodies led to co-immunoprecipitation of

the a1AT species linked to HSP70, thus confirming fusion of the proteins. The additional finding of circulating antibodies against the

HSP70-a1AT protein supported its immunogenic properties with implications for diabetes and its complications.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Diabetes mellitus; Insulin-dependent; Heat shock proteins; Heat shock protein 70; a1-Antitrypsin; Immunoglobulins; Immune complexes;

Immune markers

Heat shock proteins (HSPs) are a large group of

phylogenetically highly conserved intracellular proteins

with complex and differentiated functions [1–3]. Their

expression is significantly increased under a variety of

stressful stimuli, including pathological conditions such

as viral and bacterial infections, inflammation or au-

toimmunity [4]. Although they are generally known for

the chaperoning activity [5,6], some of these proteinsshow unique, specialized functions, both dependent on

and independent of their capacity to bind peptides and

proteins. An interesting role is that played by Grp94, the

most representative endoplasmic reticulum-resident

HSP, and by cytoplasmic HSP70, in delivering antigenic

peptides to the MHC class I processing pathway, thus

activating both humoral and cellular immune responses

[7–10]. The role of HSPs in the pathogenesis of Type 1diabetes and its complications is a still unexplored but

promising area of investigation. Recent reports have

contributed to shed light on the intriguing involvement

* Corresponding author. Fax: +39-049-827-5093.

E-mail address: paola.finotti@unipd.it (P. Finotti).

0006-291X/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2004.01.058

of both Grp94 and HSP70 in this disease. HSP70 plays a

crucial role in cellular defence against radical-dependent

damage [11]. Its up-regulation in pancreatic b-cells is

associated with improved resistance to nitric oxide tox-

icity, taken as an important mediator in b-cell destruc-tion in the experimental model of Type 1 diabetes [12].

However, it has also been observed that extracellular

HSP70 elicits a potent pro-inflammatory, cytokine-like,immune response in human monocytes [13,14]. This

result points to dual, apparently contradictory, func-

tions of HSP70, depending on whether its effects are

confined within the cell or are elicited at extracellular

level. Both Grp94 and HSP70 must be released from

damaged or necrotic cells in order to activate the im-

mune system and act as highly immunogenic carriers of

peptides to be presented to immunocompetent cells [8–10]. By acting also as a co-stimulatory, peptide-free

molecule, HSP70 represents the most immunogenic HSP

[14–16]. Continuous exposure to HSP70 and HSP70

analogues (such as those represented by various patho-

genic organisms) may challenge the immune system in

distinguishing between “self” and “non-self” [9,17–19].

298 P. Finotti, A. Pagetta / Biochemical and Biophysical Research Communications 315 (2004) 297–305

A link has been proposed to exist between HSP70 andcertain autoimmune diseases associated with MHC [19],

as testified by the finding of auto-antibodies against

HSP70 itself in patients with systemic lupus erythemat-

osus [20,21]. Recent observations that Grp94 and

HSP70 are present in high concentrations in plasma of

Type 1 diabetic patients, even at a late stage of the

disease, and are prevalently complexed with IgG [22],

prompted us to investigate in detail the nature of thisassociation, to determine whether it represents the ac-

tivation response of the immune system to the HSP

challenge.

Materials and methods

Sample collection and purification procedures. Plasma was obtained

from heparinized blood of six insulin-dependent diabetic patients (3

women and 3 men) with a mean age (�SD) of 35.6 (�9.57) years, and

of six, sex- and age-matched control subjects with a mean age (�SD) of

34.3 (�6.5) years. Mean (�SD) diabetes duration and age at onset of

diabetes were 11 (�4.9) and 24.5 (�8.6) years, respectively, whereas the

mean value (�SD) of HbA1c was 7.3 (�1.1). All subjects were non-

smokers and did not take any medication other than insulin (diabetic

subjects) nor did they show any clinical evidence of vascular and

neurological complications or other pathologies.

Plasma from both diabetic and control subjects was pooled in two

separate tubes and dialysed in a Spectrapor membrane of 15,000

MWCO (A.H. Thomas, Philadelphia, PA, USA) against a large excess

of 20mM Tris–HCl buffer solution (pH 7.4). Dialysed plasma was

diluted in the same buffer solution as above to obtain a protein con-

centration of 4–6mg/ml, before being tested for proteolytic activity on

casein. Digestion was followed in SDS–PAGE, as specified previously

[22].

Plasma purification was performed using a Con-A Sepharose 4B

(Amersham Pharmacia Biotech, Uppsala, Sweden) and an Affi-gel

Blue column (Bio-Rad, Richmond, CA, USA), both applied on a fast

liquid chromatography system (Pharmacia LKB, Uppsala, Sweden).

For plasma purification, 6.5mg plasma proteins were loaded on each

column and the eluate was monitored at 280 nm. Proteins were eluted

from the Con-A column (14ml volume) at a flow rate of 0.6ml/min,

with 20mM Tris–HCl buffered solution (with 0.5M NaCl, 1.0mM

CaCl2, and 1.0mM MgCl2, pH 7.5), in the absence (void volume) and

presence of 0.5M a-DD-methylmannoside for removal of bound pro-

teins. Elution from the Affi-gel blue column (11ml volume) was ob-

tained with a buffer solution of 50mM Tris–HCl with 0.15M NaCl

(pH 8.0) in the absence (void volume) and presence of 1.0 MNaCl, at a

flow rate of 0.8ml/min.

Fractions from both columns were submitted to ultrafiltration

(Millipore Amicon Ultra 15, Billerica, MA, USA) before being anal-

ysed further.

Immunoprecipitation. Pre-clearing was performed on dialysed and

diluted plasma samples and on HSP-enriched, proteolytically active

plasma fractions eluted from both columns. Pre-clearing was carried

out by incubating 1.0mg proteins with 75ll Protein A–Agarose CL-4B

(Sigma Chemicals, St. Louis, MO, USA) for 2 h at 4 �C, followed by

centrifugation at 1500g for 10min. The IgG-free supernatant was

submitted to the clearing step by overnight incubation with both anti-

Grp94 and anti-HSP70 antibodies (both rabbit polyclonal IgG, from

StressGen Biotechnologies, Victoria, BC, Canada). Immunocomplexes

were then pelletted by incubation with protein A–agarose followed by

centrifugation (as above, for the pre-clearing step). The pellet was

washed three times in TBS solution, each washing being followed by

centrifugation. Both the supernatant and pellet of the pre-clearing and

clearing steps were processed by electrophoresis and probed by im-

munoblotting with specific monoclonal antibodies.

Electrophoresis and immunoblotting. Electrophoresis was run at

both 10% and 12.5% polyacrylamide gel in denaturing (SDS) condi-

tions, loading 5–8lg proteins in the absence and presence of reducing

treatment. In immunoblot analysis, 5 lg proteins were transferred into

a PVDF membrane (Immobilon, Millipore, Billerica, MA, USA) and

treated in 20-ml TBS solution with anti-human a1-antitrypsin (a1-AT)

(sheep polyclonal, The Binding Site, Birmingham, UK), HSP70

(mouse monoclonal from StressGen) and Grp94 (rat monoclonal, from

StressGen) antibodies. Alkaline phosphatase conjugated affinity-puri-

fied IgGs were used as secondary antibodies. Negative controls with

secondary antibodies alone were also made to confirm the specificity of

immunostaining.

In experiments aimed at detecting antibodies in plasma from both

diabetic and normal subjects, the purified plasma fractions which

mostly contained HSPs and a1AT were processed in duplicate in SDS–

PAGE, electroblotted into a PVDF membrane, and incubated over-

night, under slow agitation, with either TBS solution alone (control

sample) or diluted plasma (both diabetic and normal) (final protein

concentration: 10–15lg/ml). After extensive washing of the membrane,

complexes formed by circulating antibodies bound to blotted proteins

were visualized in immunoblotting both directly, using primary anti-

human IgG (whole molecule) antibodies (positive visualization), and

indirectly, with anti-human a1AT antibodies (both from sheep, The

Binding Site), followed by alkaline phosphatase conjugated secondary

antibodies (donkey anti-sheep IgG, Sigma Chemicals).

Results

Separation features of plasma HSPs

To investigate the characteristics and extent of HSP

binding to plasma proteins we chose two different but

complementary affinity chromatography procedures.

Elution profiles of plasma proteins from Con-A and

Affi-gel columns appeared to be qualitatively similar,although differing significantly in amount and type of

proteins recovered in each peak (Fig. 1A). The highest

percentage of proteins (73% and 61% of those loaded in

Con-A and Affi-gel columns, respectively) was found in

the void volume, in a big peak (peak 1) comprising a

small, poorly resolved peak (pre-1) appearing as shoul-

der on the ascending branch (Fig. 1A). SDS–PAGE and

immunoblotting of peaks confirmed that most repre-sented plasma proteins, albumin, and immunoglobulins

(Igs), were separated differently by the two chromatog-

raphy procedures (Fig. 1B). Proteins in the void volume

from Affi-gel column were cleared from the bulk of al-

bumin, mostly retained into the column and only par-

tially eluting in the peak 2 even under high-salt gradient.

Igs turned out to be poorly separated by Affi-gel, ap-

pearing prevalently, but not exclusively, in peak 1. In-stead, Con-A chromatography led to better separation

of plasma Igs, almost entirely present in the peak of void

volume together with albumin (Fig. 1B).

Western blotting with anti-HSP antibodies showed

that most of plasma Grp94 and HSP70 eluted in peak 1

from both columns and partially also in peak 2 from

Affi-gel (Figs. 2 and 3). No immunoreactivity for both

Fig. 1. Elution characteristics of proteins in diabetic plasma passed

through Affi-gel and Con-A Sepharose columns. (A) Elution profiles of

6.5 mg proteins of pooled plasma loaded in each column. Protein

concentrations of peaks pre-1, 1, and 2 from Affi-gel and Con-A

Sepharose were 0.124, 0.33, and 0.09mg/ml, and 0.139, 0.575, and

0.133mg/ml, respectively. Absorbance at 280 nm is in arbitrary units.

(B) SDS–PAGE of peaks (10% acrylamide gel). Eight micrograms of

sample proteins was loaded in each well, in the absence of reducing and

heating treatments. Molecular mass markers in kilodalton on left.

Arrows indicate positions of both Igs (average molecular mass of

140 kDa) and albumin bands (65 kDa).

P. Finotti, A. Pagetta / Biochemical and Biophysical Research Communications 315 (2004) 297–305 299

HSPs was instead present in peak 2 from Con-A. While

lack of affinity for Con-A may be explained by differ-

ences in the structure of plasma HSPs with respect to

their native, intracellular counterparts, which are known

to bind to concanavalin avidly, affinity for Affi-gel, al-though poor, may be due to the fraction of HSPs bound

to albumin and eluting together under high-salt gradi-

ent. However, considering the pattern of Ig elution from

both columns, we tested the alternative possibility that

concentration of both Grp94 and HSP70 in the void

volume was simply a consequence of their stronger as-

sociation with Igs, especially IgG.

Distinct binding properties of Grp94 and HSP70 to

plasma proteins

We thus performed pre-clearing with protein A–

agarose on peak 1 from Con-A to remove the bulk ofIgG (Fig. 2A). In the supernatant, a clear positivity for

Grp94 was present in a band at about 95 kDa (monomer

of Grp94) (Fig. 2A), whereas no immunopositivity was

detected for HSP70. Both Grp94 and HSP70 were in-

stead recovered in significant amount in the pellet to-

gether with IgG (Fig. 2A). The Grp94-positive bands ofabout 100 and 120 kDa also cross-reacted with anti-IgG

antibodies (cross-reactivity especially evident in the 120-

kDa band) (Fig. 2A). Thus, Grp94 appeared to be

strongly associated with IgG even under reducing

treatment of samples in SDS–PAGE. Positivity for

HSP70 was found in bands with masses of 55, 85, 120,

and 130 kDa, fairly different from standard HSP70

which focused only in two bands at 85 and 130 kDa(Fig. 2A, right). Cross-reactivity with anti-IgG anti-

bodies was noted in the 120-kDa band of HSP70, similar

to that of Grp94 (Fig. 2A). Results indicate that the

HSPs are prevalently engaged in forming tenacious

complexes with IgG from which dissociate as free pro-

teins only partially after reducing treatment in SDS–

PAGE.

To further confirm the characteristics of Grp94 andHSP70 binding to IgG, we also submitted peak 1 from

Affi-gel to the pre-clearing step (Fig. 2B). This yielded

an overall separation of IgG-associated Grp94 and

HSP70 qualitatively similar to that seen in peak 1 from

Con-A. In the supernatant, only a faint positivity for

Grp94, but not for HSP70, was apparent at about

100 kDa, whereas in the pellet a significant immuno-

staining for Grp94 was visible in two bands at 100 and130 kDa (Fig. 2B). Positivity for HSP70 was again

present in more numerous bands, the main one of which

focused at about 110 kDa (Fig. 2B). Cross-reactivity

with anti-IgG antibodies was noted in the HSP70- and

Grp94-positive band at 100 kDa (Fig. 2B).

HSP70 is present also fused with a1AT

Pre-clearing experiments on peak 1 from both Con-A

and Affi-gel columns indicated that IgGs were the main

plasma component involved in binding both Grp94 and

HSP70. In pellets from the pre-clearing step, we also

noted positivity for a1AT, both at the expected mass of55 kDa and at higher masses, in bands cross-reacting

with anti-Grp94 (100 and 130 kDa), anti-IgG (100 kDa),

and even more intensely, with anti-HSP70 antibodies

(85, 100, and 110 kDa) (Figs. 2A and B, arrows). While

the elution of a1AT in peak 1 from Affi-gel fit its known

lack of affinity for this column (a property commonly

exploited to obtain its purification from plasma albu-

min), presence of a1AT in peak 1 from Con-A columnwas fairly unexpected, given high affinity for conca-

navalin displayed by a1AT. We thus investigated in

detail all peaks for immunoreactivity for a1AT. We

confirmed that the bulk of plasma a1AT mostly oc-

curred in peak 2 from Con-A, as expected, but it was

also present in peak 1. In both peaks from Con-A col-

umn, bands of a1AT other than the main one at 55 kDa

were visible at higher masses in proximity to the cathodewhen samples ran in non-reducing conditions in SDS–

PAGE (Fig. 3A). Under reducing treatment of samples,

Fig. 2. Partitioning of plasma HSPs and a1AT after pre-clearing of peaks 1 from both columns. (A) The supernatant and pellet of peak 1 from Con-A

column after pre-clearing were electrophoresed on 10% SDS–polyacrylamide gel and probed with the indicated antibodies in Western blotting. Five

micrograms of sample proteins was loaded in each well after reducing treatment. Molecular mass markers in kilodaltons are indicated. The large

band of albumin migrates at about 75 kDa under reducing conditions. Arrows mark the a1AT-positive bands at 55, 85, and 100 kDa. (B) The

supernatant and pellet of peak 1 from Affi-gel column after pre-clearing. Treatments of samples and other conditions as in (A). Arrows mark the

position of a1AT-positive bands at 55, 100, 110, and 130kDa. The 25-kDa band represents Ig light chains.

300 P. Finotti, A. Pagetta / Biochemical and Biophysical Research Communications 315 (2004) 297–305

the immunostaining near the cathode disappeared inboth peaks 1 and 2, whereas the 55-kDa band turned

out to be stained more intensely (data not shown). Thus,

besides being free, a1AT was also present in high-mo-

lecular mass complexes, from which it dissociates after

reduction of disulphide bridges. It is worth noting that

the high-molecular mass complexes of a1AT are not

present in the plasma of control subjects submitted to

the same chromatographic procedures (data not shown).Interestingly also, the main band of a1AT at 55 kDa in

peak 1 of Con-A column was found to immunoreact

with anti-HSP70 antibodies, a result which indicated a

close association of a1AT with HSP70 (Fig. 3A).

In peaks from Affi-gel, positivity for a1AT was con-

centrated, as expected, in peak 1, in which it appeared

not only in the 55-kDa band but also in bands at 85 and

100 kDa immunoreacting with anti-HSP70 antibodies(Fig. 3B). Moreover, in peak pre-1 from Affi-gel, a thin

band at about 53 kDa was apparent in SDS–PAGE

which immunoreacted with both anti-HSP70 and anti-

a1AT antibodies (Fig. 3B), thus mirroring the double

immunopositivity found in the similar band in peak 1

from Con-A (Fig. 3A). These results suggested that

HSP70 was closely linked to a1AT to form a fusionprotein which migrated with the same mass of normal

a1AT, but showed markedly different elution properties,

appearing in peaks from both Affi-gel (peak pre-1) and

Con-A columns (peak 1) distinct from those in which

the bulk of plasma a1AT elutes.

In order to confirm these results further, we set up

experiments of immunoprecipitation directly on a

pooled plasma sample, used as a richer source of pro-teins under examination. If the HSP70-a1AT fusion

protein also circulated free (i.e., not bound to IgG), then

it would be expected to react with anti-HSP70 and/or

anti-a1AT antibodies, forming immunocomplexes de-

tectable in the pellet after precipitation. Anti-HSP70

antibodies were preferred over anti-a1AT ones since, by

complexing the less concentrated of the two proteins,

they could yield a complete recovery of any circulatingspecies of HSP70, including that linked with a1AT. Pre-

clearing of the plasma sample with protein A–agarose

caused separation of most of IgG-linked HSP70 in the

pellet (Fig. 4). Double immunoblotting with anti-HSP70

and anti-a1AT antibodies revealed that, among the

HSP70-positive bands in the pellet, only the 55-kDa one

Fig. 3. Elution characteristics of HSP70 and a1AT from both columns.

Peaks pre-1, 1, and 2 from both Con-A and Affi-gel columns were

electrophoresed on 10% SDS–polyacrylamide gel and probed with

both monoclonal anti-HSP70 and polyclonal anti-a1AT antibodies in

Western blotting. (A) Peaks from the Con-A column. Five micrograms

of sample proteins was loaded in each well in non-reducing conditions.

(B) Peaks from the Affi-gel column. Five micrograms of sample pro-

teins was loaded in each well in reducing conditions.

P. Finotti, A. Pagetta / Biochemical and Biophysical Research Communications 315 (2004) 297–305 301

turned out to be positive for a1AT, a result which con-

firmed that the HSP70 species linked to a1AT was also

bound to IgG. Although we were unable to demonstrate

Fig. 4. Co-immunoprecipitation of HSP70 and a1AT from diabetic plasma.

protein A–agarose and supernatant and pellet were electrophoresed on 10%

polyclonal anti-a1AT antibodies in Western blotting. 0.9ml of supernatant

polyclonal antibodies and immunocomplexes were pelletted after incubation w

supernatant and pellet from the clearing step were submitted to SDS–PAG

crograms of sample proteins was loaded in each well under reducing treatment

band in the pellet of the clearing step in which both HSP70 and a1AT co-pr

any immunoreactivity for HSP70 in the supernatant ofpre-clearing (as it occurred after pre-clearing of both

peaks 1), immunoprecipitation with anti-HSP70 anti-

bodies on supernatant (clearing step) nevertheless re-

vealed in the pellet the presence of a band at 55 kDa,

positive for both HSP70 and a1AT in double immuno-

blotting (Fig. 4, arrows). This result unambiguously

demonstrated that: (i) a1AT co-precipitated with HSP70

in a single protein (both in reducing and non-reducingconditions) which became visible in the pellet only after

precipitation and concentration of immunocomplexes;

(ii) although undetectable by our methods, the fusion

protein was present in the supernatant of pre-clearing,

which proves that it circulates also free of binding to

IgG. The bulk of a1AT was not precipitated either by

protein A–agarose or anti-HSP70 antibodies, remaining,

as expected, in both supernatants of the pre-clearing andclearing steps (Fig. 4).

Anti-a1AT antibodies are present in plasma of diabetic

subjects

Since the elution profile from Affi-gel columns re-

vealed that a1AT fused with HSP70 had characteristics

markedly different from the bulk of plasma a1AT, and

considering that HSP70 fusion proteins have immuno-

logical relevance, we investigated whether HSP70-a1AT

fusion protein represents a circulating immunogen. If

this were the case, then an immune response character-

ized by production of specific auto-antibodies wouldlikely occur against either one moiety of the fusion

protein. We first chose to detect the presence of anti-

bodies against the a1AT moiety of the fusion protein by

an indirect method, in which blotted proteins of peak 1

from Affi-gel were incubated in the absence and presence

One milliliter of pooled plasma (1mg proteins) was pre-cleared with

SDS–polyacrylamide gel and probed with monoclonal anti-HSP70 and

(0.8mg proteins) was incubated overnight with 10 ll of anti-HSP70

ith protein A–agarose, as specified under Materials and methods. The

E and Western blotting as above, for the pre-clearing step. Five mi-

. Molecular mass markers are indicated in kilodalton. Arrows mark the

ecipitate.

302 P. Finotti, A. Pagetta / Biochemical and Biophysical Research Communications 315 (2004) 297–305

of diabetic (self) plasma, and then submitted to Westernblotting with anti-a1AT antibodies to detect differences,

if any, in the intensity of specific immunoprecipitation

(Fig. 5A). Similar immunostaining of the main band of

a1AT at 55 kDa was observed after incubation of blot-

ted proteins with both TBS only and plasma from

normal subjects, whereas a significant reduction in the

intensity of immunoprecipitation occurred after incu-

bation with diabetic plasma (Fig. 5A, arrow). The sameexperiment was carried out on peak 1 from Affi-gel

column of normal plasma by probing blotted proteins

with anti-a1AT antibodies after incubation with both

diabetic and normal (self) plasma. Although no change

in the specific immunostaining of a1AT was noted after

incubation with normal (self) plasma, a significant re-

duction, albeit to a lesser extent with respect to that

observed above, was visible after incubation with dia-betic plasma (Fig. 5B, arrow). This result suggested that

diabetic plasma contained anti-a1AT antibodies which

also partly recognized epitopes of normal a1AT. In or-

der to exclude that the reduction in immunostaining of

a1AT was due to aspecific binding of circulating sub-

Fig. 5. Auto-antibodies in plasma of diabetic subjects. Eight microgram p

acrylamide gel under reducing conditions, blotted, and incubated overnight w

Materials and methods. Blotted proteins were then probed in Western blott

sualization) and anti-IgG antibodies (C, positive visualization). (A,B) The ar

55 kDa band. (C) Arrows in peak 1 mark the bands of immunoprecipitati

antibodies.

stances other than auto-antibodies, which may maskantigenic sites on a1AT, we performed experiments

aimed at revealing the presence of circulating antibodies

directly. For this purpose, we first blotted peaks pre-1

and 1 from Affi-gel, and 2 from Con-A columns—these

last two peaks containing most of plasma a1AT—and

after incubation with and without diabetic plasma, de-

tection of antibodies (IgG) which bound to blotted

proteins was performed in immunoblotting with anti-IgG antibodies (positive detection) (Fig. 5C). After

subtraction of immunostaining of control lanes (incu-

bation in TBS only), bands positive for IgG, which in-

dicated the presence of auto-antibodies, were clearly

visible in peak pre-1, in the HSP70-a1AT fusion protein

at 53 kDa, whereas among numerous bands found po-

sitive in peak 1, the 55-, 85-, and 100-kDa bands were

identified as those immunoreacting with anti-a1AT- andanti-HSP70 antibodies (Fig. 5C, arrows). In peak 1, we

did not identify other proteins (possible antigens) which

reacted with circulating antibodies yielding bands of

precipitation with anti-IgG antibodies. In peak 2 from

Con-A, a single IgG-positive band was clearly shown in

roteins of indicated peaks were electrophoresed on 10% SDS–poly-

ith both normal and diabetic plasma (10lg proteins) as specified under

ing with both polyclonal anti-a1AT antibodies (A and B, negative vi-

row marks the reduction in the specific immunostaining of a1AT in the

on which also immunostained with both anti-a1AT and anti-HSP70

P. Finotti, A. Pagetta / Biochemical and Biophysical Research Communications 315 (2004) 297–305 303

correspondence of the main 55-kDa band of a1AT(Fig. 5C, arrow). Altogether, the results pointed to the

presence of circulating antibodies in the plasma of dia-

betic subjects prevalently directed against a1AT, espe-

cially that fused with HSP70.

Discussion

The aim of this investigation was to gain insight into

results of our previous work showing that in the plasma

of Type 1 diabetic subjects HSPs, mostly Grp94 and

HSP70, are present in high concentrations [22]. Specifi-

cally, we wanted to explore the nature and extent of

HSP binding to plasma proteins, in order to shed light

on the pathophysiological role of these HSPs in diabe-

tes. By using both Affi-gel and Con-A affinity chroma-tographies, we demonstrated that plasma HSPs eluted

almost entirely in association with IgG, leaving as in-

significant the part bound to albumin and IgM, which

display affinity for Cibacron-Blue of Affi-gel [23] and

concanavalin, respectively. Although we cannot exclude

that the elution of both Grp94 and HSP70 as unbound

material from both columns is also due to their intrinsic

structural characteristics, which differ from those of thenative, intracellular counterparts, our results favour the

alternative possibility that this peculiar elution property

is conferred on both HSPs by their strong binding to

circulating IgG. This conclusion is supported by the

finding that the pre-clearing step with protein A–agarose

in immunoprecipitation experiments led to the almost

entire recovery of circulating Grp94 and HSP70 in the

pellet. While the former partly escapes the binding toIgG, or alternatively, dissociates easily from IgG, also

appearing as free Grp94 in the supernatant, HSP70 is

exclusively present in plasma in complexes with IgG. In

accord with our previous results [22], we showed that

free Grp94 in the supernatant is specifically responsible

for proteolytic activity measured in this fraction (data

not shown), a finding which supports the conclusion

that HSP70 does not take part at all in eliciting thepeculiar proteolytic activity of diabetic plasma. Com-

plexation of HSPs with each other and with IgG ap-

peared to be much more intricate than it would be

expected for known circulating complexes of proteins,

since cross-reactivity with anti-IgG antibodies was

steadily observed in some Grp94- and HSP70-positive

bands even after reducing treatment of samples in SDS–

PAGE. This close association with IgG molecules isreminiscent of what occurs intracellularly, where Grp94

and other chaperones of the HSP70 family bind tran-

siently to unassembled Ig chains to stabilize their con-

formation and facilitate their correct assembly before

secretion [24]. We therefore cannot exclude that some of

the IgG-linked HSPs in diabetic plasma come from in-

tracellular compartments once the mechanisms leading

to cell damage or necrosis have taken place [7,8].However, the overall picture rather indicates that HSP-

IgG complexes represent a strong immune response

which involves both HSPs, and indirectly confirms the

complex and differentiated role played by these HSPs in

activating the immune system once they are liberated

extracellularly.

In this respect, the result showing the intriguing as-

sociation of HSP70 with plasma a1AT is of particularinterest. Although we previously observed, and here also

confirmed, that Grp94 in plasma can form complexes

with a1AT [22], our present results reveal different

pathophysiological implications for the association of

HSP70 with a1AT. In Affi-gel chromatography, it was

apparent that HSP70 eluting early, just before the bulk

of HSP70 present in the main peak of the void volume,

cross-reacted with anti-a1AT antibodies and was visibleonly in a band of the expected mass of a1AT, indicating

the presence of a HSP70-a1AT fusion protein. This was

definitively confirmed by experiments of immunopre-

cipitation with anti-HSP70 antibodies performed on a

plasma sample, showing co-immunoprecipitation of

a1AT with HSP70 in the pellet. Furthermore, the ob-

servation that after any pre-clearing step (on the void

volume from both columns and on plasma samples) theresulting pellet always contained a1AT, mostly detected

in bands which also immunostained for HSP70, strongly

suggests that protein A-precipitable, IgG-linked a1AT is

only that associated with HSPs, especially HSP70. In-

deed, the bulk of plasma a1AT, which was found in both

peak 1 from Affi-gel and peak 2 from Con-A columns, in

accord with the known elution characteristics of normal

a1AT [25], was neither precipitated by protein A–aga-rose nor did it react with anti-HSP70 antibodies in im-

munoprecipitation experiments. These results suggest

that the species of a1AT linked with HSP70 circulate(s)

as immunocomplexes.

It is known that among HSPs, HSP70 plays an exqui-

site role in stimulating the immune system, both in pep-

tide-dependent and -independent manners [10,14,16].

Thus, in addition to delivering non-covalently boundpeptides to the MHC class-I pathway, and activating

cytotoxic T-lymphocytes (CTL) efficiently, it can also

deliver stimulatory signals directly on cells of the innate

immune system [15,16], or even on cells which do not

belong to the immune system, but can interplay with it [9].

Moreover, the covalent conjugation of antigenic proteins

and peptides to HSP70 in generating fusion proteins can

elicit a potent humoral and CTL-mediated immune re-sponse directed against the fusion partner [17,26–28],

which also appears to be independent of the chaperone

property of the HSP [28]. Based on these and other nu-

merous reports stressing the immunogenic properties of

HSP70 and HSP70-carried peptide/proteins [29–32], we

investigated whether the HSP70-a1AT fusion protein was

actually an immunogen. To prove this, we tested plasma

304 P. Finotti, A. Pagetta / Biochemical and Biophysical Research Communications 315 (2004) 297–305

for the presence of auto-antibodies directed against thefusion partner, i.e., a1AT moiety. Experiments in which

blotted peaks containing most of plasma a1AT were in-

cubated with both normal and diabetic (self) plasma,

before being probed with anti-a1AT antibodies, showed

that immunoprecipitation in the main band of a1AT was

significantly reduced only after incubation with diabetic

(self) plasma. The result clearly indicated that, after in-

cubation with diabetic plasma, antigenic sites of blotteda1AT were partially masked, being thus reduced their

overall availability to react further with anti-a1AT anti-

bodies in immunoblotting. Confirmation that this effect

was actually due to the specific binding of circulating anti-

a1AT antibodies came from experiments in which blotted

proteins of peaks, after incubation with both diabetic and

normal plasma, were probed in immunoblotting with

anti-human IgG antibodies: specific immunostaining forIgG, not present in the control, i.e., in the absence of di-

abetic plasma, was visible in bands which also immuno-

stained for both a1AT and HSP70. We also showed that

circulating anti-a1AT antibodies were partly directed

even against normal a1AT, as proved by: (i) the reduction

of a1AT immunostaining in peak 1 from Affi-gel column

of normal plasma, after incubation with diabetic (non-

self) plasma and (ii) the presence of antibodies precipi-tating on the main band of a1AT in peak 2 of Con-A after

incubation with diabetic (self) plasma.

Besides proving that the HSP70-a1AT fusion protein

actually has the characteristics of an immunogen, our

results also indicate that a continuous immunogenic

stimulation may have caused an expansion of the im-

mune response with the production of antibodies cross-

reacting with normal epitopes on the a1AT molecule.Further studies are necessary to establish the role of

HSPs in general, and HSP70 fusion proteins in partic-

ular, in the pathogenesis of diabetes and/or its compli-

cations. The first important questions to be addressed

are as to whether this circulating HSP70 fusion protein

may appear early in the course of disease, thus repre-

senting a marker of the diseases itself, and what its

source may be once the processes leading to b-cell de-struction have been completed (as is the case in subjects

analysed in this work).

Acknowledgments

We thank Professor G.A. Danieli for his helpful discussion of the

manuscript. This work was supported by MURST (Ministero

dell’Universit�a e della Ricerca Scientifica e Tecnologica) grants of ex-

60%.

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