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Duchenne dystrophy Focal alterations in the distribution of concanavalin a binding sites at the muscle cell surface.

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Duchenne Dystrophy: Focal Alterations
in the Distribution of Concanavalin A
Binding Sites at the Muscle Cell Surface
Eduardo Bonilla, MD, Donald L. Schotland, MD, and Yoshihiro Wakayama, M D
In 6 patients with Duchenne dystrophy, electron microscopical studies of concanavalin A (Con A) binding sites using
the peroxidase labeling technique revealed a population of muscle fibers in which the reaction at the cell surface was
irregular and patchy in areas larger than 0.5 p. In 5 control subjects, such focal alterations were not observed and the
reaction appeared continuous and regular. The findings indicate focal abnormalities at the muscle cell surface of Con
A receptors in Duchenne dystrophy.
Bonilla E, Schotland DL, Wakayama Y: Duchenne dystrophy: focal alterations in the distribution of
concanavalin A binding sites at the muscle cell surface. Ann Neurol 4 : 117-123, 1978
lamina, as previously proposed by Bennett [2, 31.
T h e results show focal alterations at the cell surface
in the distribution of Con A receptors in a population
of muscle fibers from patients with Duchenne dystrophy.
Electron microscopical studies with peroxidaselabeled lectins have been used recently for the detection of cell surface carbohydrates of both normal and
transformed cells [ 4 , 6 , lo]. Concanavalin A (Con A),
a protein isolated from jack bean, reacts specifically
with terminal a-D-mannose, a-D-glucose, and a-Dfructose of carbohydrate chains [ 8 , 9 ] . Con A binding
sites can be visualized using peroxidase as a marker
which, in the presence of 3'-3'-diaminobenzidine and
hydrogen peroxide, gives an electron-dense reaction
product [4].Recent Con A peroxidase binding studies of in situ preparations of skeletal muscle and isolated muscle plasma membrane have shown that Con
A binds to both the plasma membrane and the basal
lamina [ 11.
Several lines of evidence have implicated a muscle
cell surface abnormality in Duchenne dystrophy.
Blood concentrations of muscle enzymes are elevated [ 7 ] ,possibly d u e to a defective muscle cell surface which allows the penetration of large molecules
such as peroxidase [ 151; activation of adenyl cyclase
by epinephrine is abnormal, possibly due to a defective receptor at the muscle cell surface [ 13, 141; and
recent freeze-fracture work has shown an alteration
in the internal molecular architecture of Duchenne
muscle plasma membrane 1181.
The aim of this investigation was to study the muscle cell surface in patients with Duchenne dystrophy
using the Con A peroxidase labeling technique.
Throughout this report, the term crllsurfuce is used t o
encompass both the plasma membrane and the basal
Materials a n d Methods
Muscle biopsies were obtained from 6 patients with
Duchenne dystrophy. Progressive proximal weakness and
enlarged calves had been present since early childhood. All
had markedly elevated creatine phosphokinase, in excess of
1,000 IU per liter, and muscle biopsies consistent with this
disorder. Four had evidence of X-linked recessive inheritance. The age at biopsy was between 4 and 10 years.
For control data, normal human muscle was obtained
from 5 individuals (1 adult male volunteer without
neuromuscular disease, 2 nonweak female and male controls, and 2 male children aged 11 weeks and 5 years who
were undergoing orthopedic operations). Histochemical
studies were normal. For all biopsies an informed consent
was obtained.
The specimens were removed at rest length in a
U-shaped muscle clamp or attached to a stick and fixed
immediately in 3% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 for 20 minutes. Strips of muscle were carefully dissected o u t and stored for 12 hours at 4°C in Karnovsky fixative. The samples were incubated after fixation
in a solution of 0.1 M lysine at pH 7.4 for 1 hour at room
temperature. This step was performed to inactivate free
aldehyde groups of glutaraldehyde capable of binding free
amino groups of Con A. Then the material was washed for
four days in several changes of 0.2 M phosphate buffer, pH
7.4. Under the dissecting microscope, 5 to 8 fiascicles were
-
From the Henry M. Watts Jr. Neuromuscular Disease Research
Center and the Department of Neurology, University of Prnnsylvania School of Medicine, Philadelphia, PA.
Address reprint requests to Dr Bonilla, Department of Nrurology, Hospital of the University of Pennsylvania, Philadelphia, PA
19104.
Accepted for publication Feb LO, 1978.
0364-5134/78/0004-0204$01.25 @ 1078 by Eduardo Bonilla
117
dissected out, and one tissue block (1 to 2 mm thick) from
each fascicle was sampled. The method of Con A labeling
of carbohydrates was that of Bernhard and Avrameas as
outlined by Bretton and Bariety [ 4 , 51. All steps were carried out with constant gentle agitation at room temperature, and the blocks were incubated in the following manner: (1) crystallized and lyophilized Con A (Miles Laboratory) at a concentration of 100 pg/ml in 0.2 M phosphate
buffer at pH 7.4 for 4 hours, then washed overnight' with
five changes of phosphate buffer; (2) peroxidase at a concentration of 100 pg/ml (Sigma type VI) in 0.2 M phosphate buffer, p H 7 . 4 , for 3 hours, followed by a 2 hour
wash in five changes of buffer; and (3) 3'-3'-diaminobenzidine, 0.5 mg/ml, in 0.2 M Tris buffer at p H
7.4 (containing 0.02% H,O,) for 15 minutes. Control tissue blocks were incubated with Con A in the presence of
0.2 M a-methyl-D-mannoside, an inhibitor of Con A binding to sugars [ 4 , 51. In order to achieve almost complete
inhibition, Con A and tissue blocks were incubated separately for l hour at 37°C with the inhibitor and then were
incubated together at room temperature for 4 hours [ 101.
All samples were postfixed for 1 hour in 2% osmium tetroxide in phosphate buffer at p H 7.2, dehydrated in serial
alcohols, and embedded in Epon.
The embedded material was sectioned with a glass knife
mounted on an LKB ultramicrotome. Cross-sections 0.5 p
thick were mounted on glass slides and examined without
118
Annals of Neurology
Vol 4
No 2 August 1978
stain under a Zeiss I1 microscope. Muscle fibers with a
complete layer of brownish material at the cell surface were
counted and then sectioned thin with a diamond knife. The
sections were stained with lead citrate for 5 minutes, examined, and photographed in an AEI EM-6B electron microscope operating at 60 kv. A minimum of 20 cross-sectioned
fibers were studied for each patient and control.
Results
A total of 112 muscle fibers from the 5 control subjects were studied. The cell surface was coated by a
regular and continuous layer of electron-dense material, ranging in width from 600 to 1,000 A, which
covered the plasma membrane and basal lamina (Fig
1, Table). The caveolae were also heavily stained. No
difference in the pattern of the reaction was noted
between children and adults. The reaction was virtually absent in control specimens incubated with 0.2
M a-methyl-D-mannoside (Fig 2).
A total of 129 muscle fibers from the 6 patients
F i g I. Con A peroxidu~ereaction in normal human muscle. The
cell .rugace is coated by a continuous a n d regular layer of
electron-dense material. Reaction product is also seen within the
raveokae. ( X26,250.)
Concanavalin A Binding i n Normal Subjects a n d in Patients with Duchenne Dystrophy
No. of Muscle Fibers
Showing Reaction"
Subjects
Normal subjects ( N = 5)
Patients with Duchenne
dystrophy ( N = 6)
112
129
-
Findings on Electron Microscopy"
Normal Reaction
Abnormal Reaction
112 (100%)
87 (67%)
42 ( 3 3 % )
0
"Presence of a complete layer of brownish material surrounding the fiber at high-resolution light microscopy (semithin, unstained sections).
bobsemations were based on examination of transversely cut fibers in a single plane of sectioning. Normal reaction: complete layer of
electrondense material at the cell surface; abnormal reaction: patchy reaction in focal areas larger than 0.5 p.
with Duchenne dystrophy were studied. In 87 muscle fibers the reaction did not appear different from
that observed in the controls, but in 42 fibers the
reaction was patchy and irregular in areas larger than
0.5 p (Fig 3), in sharp contrast to the observations
made in controls.
A sequential study of the focal abnormalities of
Con A binding at the cell surface of these fibers suggested two early lesions characterized by loss of regularity of the reaction pattern. I n one, the plasma
membrane was still preserved (Fig 4A,B); in the
other, the plasma membrane appeared disrupted (Fig
4A,C). These areas were associated with a focal inF i g 2. Normal human muscle exposed to Con A i n the presence of
0.2 M a-methykwnannoside. The reaction a t the cellsurface is nearly absent. (X26,250.)
crease in extracellular collagen. The intracellular
structures underlying these early focal defects appeared normal, without ultrastructural features of
either degeneration o r regeneration. A more advanced focal lesion showed interruptions in the regular pattern of the reaction associated with focal
penetration of Con A peroxidase. The intracellular
structures underlying these defects showed rarefaction of contractile proteins and dilatation of the sarcotubular system (Fig 4D). A third or late focal lesion
showed interruptions in the regular pattern of the
reaction at the cell surface with focal penetration of
Con A peroxidase, and the intracellular structures
underlying these focal defects disclosed profound
ultrastructural changes (see Fig 3). Examination of
control blocks from dystrophic patients incubated
with 0.2 M a-methyl-D-mannoside revealed that the
Bonilla, Schotland, and Wakayama: Con A Binding Sites in Duchenne Dystrophy
119
F i x 3. Con A pervxihse recritioti in Duchennedystrophy. Portions of two niu.rilejibersare shouw. The reaction i n thejiberat
the upper portion ofthe niiiro,qraphappears continuous und
re.gular. Thefiber itr the lou~erpdrt of the niicrograph shoiiv crn
120 Annals of Neurology
Vol 4
No 2
August 1978
irreKular anddiscontinuous pattern of reaction at the cell surfare. I n addition, there is focalpenetration of Con A peroxidate
( arrows ) and u ItraJ-tru rtu ral alterat io ris of int racellular co mpotients. i~ l j . 0 0 before
0
>%reduction.)
F i g 4. Con A peroxidtrse reaction in Duchenne dy.rtrophy. ( A )
E d y focal hions, marked by bars. The one i n the right portion
of the micrograph hwJpre.rervation of the plasma membrane;
the one on the left shou1.r disruption of the membrane. The intracellular structures underlying both focal defects appear normal. ( ~ 2 5 , 0 0 0 .()B ) HiKher magnification of theficaldefect
a.r.rociatedwithpreservation of the plctsma membrane (shown on
the right side in A). (X35,OOO.) (C)Higher magnification of
thefocal defect associated with disruption of the plasma membrzzne (shown on the left side in A). (X35,OOO.) (0)
Advanced
lesion, showing interruptions i n the regular pattern of the reaction associated with focal penetration of Con A peroxidase (arrows). I n addition, the intracellular structures underlying the
defect show rarefaction of contractile elements and dilatation of
the sarcotubular system. (X25,OOO. All before 5 96reduction.)
reaction at the cell surface was virtually absent, as in
the normal controls (see Fig 2).
When the fibers completely surrounded by Con A
peroxidase reaction and those with focal alterations
larger than 0.5 p were counted for the 6 dystrophic
patients, 33% of the fibers examined showed focal
alterations in Con A binding at the cell surface (see
the Table). In the controls, such focal alterations
were not seen. The difference between the dystrophic patients and controls was significant.
Discussion
The results of this study show focal alterations in Con
A binding at the cell surface of a population of muscle fibers from patients with Duchenne dystrophy.
This contrasts with the regular and continuous pattern of Con A binding observed in normal individuals. The findings indicate focal abnormalities at the
muscle cell surface of Con A receptors in Duchenne
dystrophy, suggesting an alteration in the distribution
of a-D-mannose, a-D-glucose, and a-D-frUCtOSe since
Con A binds specifically to these carbohydrate residues [4-6, 8-10, 161.
Directly relevant to the results of these investigations are previous combined ultrastructural and electron cytochemical studies in Duchenne dystrophy.
Recently, Mokri and Engel [IS] demonstrated a
population of nonnecrotic muscle fibers with focal
absence or disruption of the surface membrane. Underlying these focal defects were structural abnormalities of intracellular components. In addition,
penetration of peroxidase into the focal lesions suggested an abnormality of muscle fiber permeability
consonant with the hyperenzymemia of the patients
[15]. I t is possible that the Duchenne muscle fibers
with focal alterations in Con A binding described in
this study may correspond to the fibers reported by
Mokri and Engel, since we noted similar ultrastructural alterations in areas underlying the focal defects in association with penetration of the Con A
peroxidase complex (see Figs 3, 4D). Moreover, the
early focal defects in Con A binding noted in association with intact plasma membrane and normal underlying intracellular structures in Duchenne muscle
fibers may represent an abnormality that precedes
disruption of the cell surface membrane and subsequent formation of delta lesions [IS].
The possibility that muscle fiber regeneration may
be associated with focal alterations in Con A binding
at the cell surface must also be considered. However,
the early focal defects in Con A binding noted in this
study were not associated with underlying ultrastructural features of regeneration. In addition, the sequential study of focal alterations of Con A binding
in Duchenne muscle fibers suggested a progressive
deterioration of muscle fiber structure.
122
Annals of Neurology Vol 4 No 2 August 1978
The presence of focal alterations in Con A binding
in a population of muscle fibers from patients with
Duchenne dystrophy raises interesting questions. Do
muscle satellite cells have the same focal defects in
Con A binding? Do the carriers of Duchenne dystrophy have similar focal defects? Further Con A
binding studies are being carried out in conjunction
with examination of other neuromuscular disorders
in an effort to answer these questions.
If focal alterations at the muscle cell surface are
important in the pathogenesis of Duchenne
dystrophy-and
several investigations have suggested that they are [15, 17, 181-it would also be of
interest to know whether focal alterations in Con A
binding are fully repaired. This question focuses on
the problem of muscle regeneration in Duchenne
dystrophy. Previous studies of muscle regeneration
in the disorder have suggested that attempts to regenerate occur but ultimately fail [11, 121. Our sequential study of the focal defects in Con A binding
suggested progressive deterioration of muscle fibers.
These observations raise the possibility that attempts
to repair focal defects in the distribution of Con A
receptors may also fail. If this is the case, then the
early focal alteration in Con A binding noted in the
sequential study may be playing an important role in
the pathogenesis of cell death in Duchenne dystrophy.
Recent freeze-fracture studies from patients with
Duchenne dystrophy have shown nonuniform distribution and depletion of intramembranous particles
at the muscle cell surface membrane [18]. Whether
some of the intramembranous particles (as seen with
the freeze-fracture technique) in skeletal muscle
plasma membrane bear a relationship to Con A receptors (as seen with the Con A peroxidase labeling
technique) is uncertain and remains to be established.
Focal alterations in the distribution of Con A binding sites in Duchenne dystrophy add one more feature to the growing list of work implicating a muscle
cell surface abnormality in the disorder.
Supported by a Research Center Grant from the Muscular Dystrophy Association and by Grants NS-08075 and 5MOlRR00040
from the US Public Health Service.
We thank Dr Nicholas K. Gonatas for helpful discussions and
advice. We also thank Mrs Margaret Van Meter and Miss Alice
McCaughin for skillful technical assistance.
References
1. Barchi RL,Bonilla E, Wong M: Isolation and characterization
of muscle membranes using surface-specific labels. Proc Natl
Acad Sci USA 74:34-38, 1977
2. Bennett HS: Morphological aspects of extracellular polysaccharides. J Histochem Cytochem 11:14-23, 1963
3. Bennett HS: Cell surface: components and configurations, in
Lima-de-Faria A (ed): Handbook of Molecular Cytology.
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Bernhard W, Avrameas SA: Ultrastructural visualization of
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Exp Cell Res 64:232-236, 197 1
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Bretron R. Wicker R, Bernhard W: Ultrastructural localization of concanavalin A receptors in normal and SV40 transformed hamster and rat cells. Int J Cancer 10397-410, 1972
Dreyfus JC, Shapira G , Schapira F: Biochemical study of muscle in progressive muscular dystrophy. J Clin Invest 33:794797, 1954
Goldstein IJ, Hollerman CE, Merrick JM: Proteincarbohydrate interaction: I. The interaction of polysaccharides with concanavalin A. Biochem Biophys Acta
97:68-76, 1965
Goldstein IJ, So LL: Protein-carbohydrate interaction: 111.
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11. Hudson P, Pearce GW, Walton JN: Preclinical muscular dystrophy: histopathological changes observed on muscle biopsy.
Brain 90:565-576, 1967
12. Mastaglia FL, Papadimitriou JM, Kakulas BA: Regeneration
of muscle in Duchenne dystrophy. J Neurol Sci 11:425-444,
1970
13. Mawatari S, Miranda A, Rowland LP: Adenyl cyclase abnormality in Duchenne muscular dystrophy: muscle cell in culture. Neurology (Minneap) 26:1021-1026, 1976
14. Mawatari S, Takagi A, Rowland LP: Adenyl cyclase in normal and pathologic human muscle. Arch Neurol 30:96-102,
1074
15. Mokri B, Engel AG: Duchenne dystrophy: electron microscopic findings pointing to a basic or early abnormality in the
plasma membrane of the muscle fiber. Neurology (Minneap)
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16. Nicholson GS: Topography of membrane concanavalin
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17. Schmalbruch H: Segmental fiber breakdown and defects of
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Bonilla, Schotland, and Wakayama: Con A Binding Sites in Duchenne Dystrophy
123
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site, muscle, distributions, focal, duchenne, surface, concanavalin, alteration, binding, cells, dystrophy
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