ARTICLE |
Correspondence to: Antonio Raggi, Dipartimento di Scienze dell’Uomo e dell’Ambiente, Chimica e Biochimica Medica, Università di Pisa, via Roma 55, 56126 Pisa, Italy.
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Summary |
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Histidineproline-rich glycoprotein (HPRG) is a protein that is synthesized by parenchimal liver cells. The protein has been implicated in a number of plasma-specific processes, including blood coagulation and fibrinolysis. We have recently reported the association of an HPRG-like protein with rabbit skeletal muscle AMP deaminase (AMPD). The results of the immunological analysis reported here demonstrate that an antibody against human plasma HPRG reacts with an AMPD preparation from human skeletal muscle. To probe the localization of the putative HPRG-like protein in human skeletal muscle, serial sections from frozen biopsy specimens were processed for immunohistochemical and histoenzymatic stains. A selective binding of the anti-HPRG antibody to Type IIB muscle fibers was detected, suggesting a preferential association of the novel protein to the AMPD isoenzyme contained in the fast-twitch glycolytic fibers. (J Histochem Cytochem 47:255260, 1999)
Key Words: histidineproline-rich glycoprotein, AMP deaminase, human skeletal muscle, Western blot analysis, immunohistochemical analysis, histoenzymatic analysis
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Introduction |
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Histidineproline-rich glycoprotein (HPRG) is a protein present at a relatively high concentration in the plasma of vertebrates. Its specific function remains unclear, although it has been implicated in several phenomena, including blood coagulation and fibrinolysis (
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Materials and Methods |
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Collection of Samples
Eleven biopsy specimens from deltoid (n = 6), quadriceps femoris (n = 3), and tibialis anterior (n = 2) were selected among frozen muscle samples collected at the Division of Pathology, Department of Oncology, University of Pisa. Eight-µm serial sections were cut in a cryostat at -20C. Biopsy of human quadriceps femoris used for AMPD preparation was performed on a patient who underwent surgery for diagnostic purposes.
On examination, no histological or histochemical abnormalities were present in all the biopsies used. All the procedures followed were approved by the University Hospital Ethics Committee, Pisa, Italy.
Preparation of AMPD from Human Skeletal Muscle
AMPD was prepared from 2 g quadriceps femoris by the procedure previously described for the isolation of the enzyme from rabbit red muscle (
Synthesis of the HPRG Peptide and Preparation of a Polyclonal Antibody to Human Plasma HPRG
A synthetic peptide equivalent to residues 462471 of human plasma HPRG was synthesized at the Krebs Synthesis & Sequencing Facility at the University of Sheffield, UK. Synthesis was performed on a Milligen 9050 Peptide Synthesizer using standard F-MOC active ester chemistry. The cleaved product was purified by HPLC using a Vydac C18 HPLC column (250 x 22 cm) (Phenomenex; Torrance, CA) and was validated by sequence determination and by mass spectrometry. Cysteine was incorporated at the C-terminus to allow conjugation of the peptide to porcine thyroglobulin using MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester) (Pierce; Rockford, IL).
A polyclonal antibody to human plasma HPRG was raised in a female New Zealand White rabbit by using the conjugated synthetic peptide. The antigen (200 µg) was emulsified in Freund's complete adjuvant (Sigma; St Louis, MO) and injected into multiple intradermal sites distributed over the back of the shaved rabbit. Six weeks later, a second injection of the antigen (50 µg) emulsified in Freund's incomplete adjuvant (Sigma) was carried out as described above, and after 4 weeks the animal was boosted with 50 µg of soluble antigen injected IV. Three months after the first injection, the animal was sacrified and exsanguinated and the serum was prepared, aliquotted, and stored at -20C. The IgG isolation from the rabbit antiserum was performed as described by
Dot-blot, SDS-PAGE, and Western Blot Analyses
One µg, 100 ng, and 10 ng of the HPRG synthetic peptide and 5 µl of human plasma were spotted on a nitrocellulose membrane (Sigma) and air-dried. After a blocking step with 3% (w/v) BSA0.5% (v/v) Tween-20 in PBS for 1 hr, the blot was washed once with PBS and then incubated with the rabbit anti-HPRG polyclonal antibody [1:10000 (v/v) in PBS] for 1 hr. A washing step with PBS was then followed by the incubation with a horseradish peroxidase-labeled goat anti-rabbit IgG (Sigma) for 1 hr [1:10000 (v/v) in PBS containing 0.01% (w/v) SDS]. The blot was extensively washed with 0.5% Tween-20 in PBS and the peroxidase reaction was developed using 3,3'-diaminobenzidine tetrahydrochloride (Sigma) as substrate. All the incubations were performed at room temperature (RT).
Electrophoresis in the presence of 0.1% (w/v) SDS was carried out on a 10% (w/v) polyacrylamide slab gel in 0.1 M Tris/0.1 M Bicine (pH 8.3). Prestained molecular weight standards (Bio-Rad; Richmond, CA) were used to calibrate the gel. Samples were run in duplicate for Coomassie Brilliant Blue staining and Western blot analysis.
After the SDS-PAGE, samples were electrotransferred to a nitrocellulose membrane. The blot was blocked with 3% BSA in PBS for 2 hr and then treated with the rabbit anti-HPRG polyclonal antibody diluted 1:10,000 (v/v) with PBS (overnight incubation at 4C). After washing with PBS, the blot was incubated with [125I]-protein A (Amersham; Sunnyvale, CA) for 2.5 hr at RT and then extensively washed with 0.5% Tween-20 in PBS and air-dried before being processed for autoradiography.
Histochemistry
Serial sections from muscle biopsies were processed for the following histological and histochemical stains: hematoxylineosin, NADH tetrazolium reductase (NADH-TR), routine ATPase (pH 9.4), ATPase preincubated at pH 4.3 and 4.6 (
Immunohistochemistry
Slides were fixed in cold acetone/methanol (1:1, v/v) for 10 min at -20C and then washed with PBS for 10 min at RT. After a 30-min blocking step for endogenous peroxidase activity with 3% (w/w) H2O2 in methanol at RT, slides were washed with PBS and incubated with the rabbit anti-HPRG primary antibody [1:100 (v/v) in PBS containing 0.5% BSA and 0.2% (w/v) gelatin] for 1 hr at 37C. As a second antibody, a horseradish peroxidase-labeled goat anti-rabbit IgG was used, diluted 1:250 (v/v) in PBS containing 0.5% BSA and 0.2% gelatin (1 hr at RT). The peroxidase reaction was developed using 3,3'-diaminobenzidine tetrahydrochloride as substrate. Controls included omission of primary antibody and substitution of immune serum by preimmune or nonimmune serum.
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Results |
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Antiserum Reactivity with Human Plasma HPRG
The reactivity of the IgG fraction isolated from rabbit antiserum was first checked by dot-blot analysis. A clear positive reaction was observed against human plasma and the HPRG synthetic peptide utilized as antigen for the immunization. No positive reaction was ob-tained when preimmune or nonimmune serum was used (not shown). Moreover, when human plasma was tested by Western blot analysis, a clear immunoreactive band at a molecular mass of approximately 50 kD was found. Reactivity of small amounts of more slowly migrating material (5575 kD) was also observed (Figure 1, Lane 4).
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SDS-PAGE and Western Blot Analysis of Human Skeletal Muscle AMPD
In a recent article we demonstrated the association of a HPRG-like protein to rabbit skeletal muscle AMPD (
Histochemistry and Immunohistochemistry
Evaluation of muscle biopsies with NADH-TR histochemical staining enabled us to classify fibers as Type I (slow-twitch) and Type II (fast-twitch). Type II fibers varied from 30 to 65% among samples. Type I fibers appeared darkly stained, whereas Type II fibers were characterized by a pale gray cytoplasm with a diffuse blue granularity that, on the basis of its density, enabled us to distinguish two subtypes, light and intermediate, corresponding to IIB (fast-twitch glycolytic) and IIA (fast-twitch oxidativeglycolytic) fibers, respectively (Figure 2A, Case A). The fiber typing obtained with NADH-TR was confirmed by the ATPase staining after acid or alkaline preincubation (not shown).
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Figure 2B shows a serial cross-section of Case A muscle biopsy immunostained for HPRG as described in Materials and Methods. All fibers showed a brown subsarcolemmal staining, but two fiber-types could be distinguished according to the sarcoplasmic shading (light and dark). The light fibers showed a granular positivity and a surrounding pale sarcoplasm, whereas dark fibers exhibited a similar granular pattern on a darker sarcoplasmic background. Comparison of this specimen with the serial section stained for NADH-TR (Figure 2A) showed a correspondence between the darker immunostained fibers and the IIB fiber type, whereas the lightly immunostained fibers corresponded to the I plus IIA fiber types.
The above observations indicate the presence of an HPRG-like protein in the glycolytic fibers, which are well known to contain the highest level of AMPD among muscle fibers (
With the HPRG immunostaining, in one of the 11 cases examined (Case B, Figure 2D) three different fiber types were detectable (light, intermediate, and dark). The light and intermediate fibers, with a similar cytoplasmic reticular network, differed in intensity of the sarcoplasmic staining. The dark fibers, which exhibited a cytoplasmic shading that was similar to that of the intermediate fibers, could be identified by a pronounced network, related to a coarser stain thickening. When a serial cross-section of the same case was stained for AMPD (Figure 2E), only two fiber types were detectable, corresponding to Types I and II described by
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Discussion |
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HPRG is a glycoprotein member of the cystatin superfamily that is synthesized by parenchymal liver cells (
Immunoblot analysis demonstrated that the anti-HPRG antibody reacts with a plasma component with an apparent molecular mass of 50 kD. This value is in agreement with the molecular weight calculated for human plasma HPRG free of carbohydrate on the basis of its amino acid composition (
When the Western blot analysis was performed using freshly prepared human skeletal muscle AMPD, a clear immunoreactivity was detected, thereby extending to the human enzyme the association with a HPRG-like molecule, first observed for rabbit AMPD (
By combining the data of our immunohistochemical and histoenzymatic analyses, a clear indication is obtained of a preferential binding of the anti-HPRG antibody to Type IIB muscle fibers. This observation deserves consideration in the light of our recent finding (
As reported by
Further studies are necessary to establish whether the novel protein participates in the structure of skeletal muscle AMPD or simply binds to the holoenzyme. However, attempts to define the physiological function of muscle HPRG should take into account the possible role of white muscle AMPD in regulating the relative concentrations of adenine nucleotides during sustained contractile activity (
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Acknowledgments |
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Supported by a grant from the Italian MURST.
We thank Mr Stefano Mazzoni for assistance in rabbit immunization and Mr Piero Bertelli for skilled technical assistance.
Received for publication June 5, 1998; accepted October 13, 1998.
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