Scand. J. Immunol.

54, 404±408, 2001

Shoulder-Pad Sign of Amyloidosis: Structure of an Ig Kappa III

Protein
J. J. LIEPNIEKS,* C. BURT* & M. D. BENSON*²
*Department of Pathology & Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana; and ²Richard L. Roudebush
Veteran Affairs Medical Center, Indianapolis, Indiana, USA

(Received 5 March 2001; Accepted in revised form 10 May 2001)

Liepnieks JJ, Burt C, Benson MD. Shoulder-Pad Sign of Amyloidosis: Structure of an Ig Kappa III Protein.
Scand J Immunol 2001;54:404±408
While amyloid infiltration of articular structures is rare, the `shoulder-pad' sign resulting from periarticular
soft tissue amyloid deposition is essentially pathognomonic for immunogloblin (Ig) (AL) amyloidosis. We
report the characterization of an amyloid protein (GRA) which produced articular amyloid deposits and the
shoulder-pad sign. Amyloid fibrils were isolated from soft tissue shoulder mass of a patient with systemic
AL amyloidosis. The fibrils were solubilized in guanidine HCl and proteins separated by Sepharose
chromatography. Amino acid sequence of fractionated protein was determined after tryptic digestion.
Sequence analysis of the major amyloid protein yielded a kappa III Ig light chain structure. The entire
variable region (VL) plus the constant region (CL) to residue 207 was identified; but lesser amounts of CL
than VL were present. A number of amino acid residues previously not observed in kappa III VL proteins,
plus a two amino acid insert (95A, 95B), were identified. Kappa III VL amyloid proteins are rare and may
show an increased predilection for soft tissue deposition. While several unique amino acid residues that were
identified in protein GRA may contribute to soft tissue amyloid deposition, no definite pattern is obvious
from comparison with other reported kappa III amyloid proteins.
Dr M. D. Benson, Department of Pathology & Laboratory Medicine, Indiana University School of Medicine,
975 West Walnut Street, #IB-503, Indianapolis, IN 46202±5121, USA. E-mail: mdbenson@iupui.edu

INTRODUCTION of articular or bony structures and visceral involvement is much

less common [5]. The b2-microglobulin protein, which is
Massive amyloid infiltration of articular structures is a rare
derived from the ancestral Ig domain gene, shares considerable
(, 2%), but well recognized manifestation of Ig (AL)
structure with that of the Ig light chain protein [6,7]. It would
amyloidosis [1,2]. As with macroglossia, which is much more
therefore be rational to hypothesize that structural determinants
common (approximately 10%), the `shoulder-pad' sign owing to
of certain Ig light chain proteins might explain their predilection
periarticular soft tissue amyloid deposition, is considered
for involvement of articular structures. Here we report the
essentially pathognomonic for AL amyloidosis. Amyloid
primary structure of an AL protein isolated from amyloid
infiltration of the soft tissues around the hip joints and in the
resected from the shoulder of a patient with the classic
femoral head may also be seen in patients with this syndrome,
`shoulder-pad' sign. The kappa III structure is analyzed in
but is less frequently reported. This rare manifestation of AL
relation to other reported proteins of this subclass which have
amyloidosis is usually part of the systemic process which
been isolated from similar tissues.
involves other organ systems such as the heart and kidneys [2].
Although articular amyloid may be associated with significant
pain and functional disability, it is the visceral involvement with
amyloidosis that usually is the cause of death [3,4]. MATERIALS AND METHODS
The reason for articular involvement with AL amyloid is not The patient. The patient, GRA, was a 75-year-old Caucasian man
clear. In the case of b2-microglobulin amyloidosis related to who presented with a 1-year history of enlargement of his shoulders
chronic renal dialysis, the majority of patients have involvement with decreased range of motion. During this time he also developed

q 2001 Blackwell Science Ltd


symptoms of bilateral carpal tunnel syndrome and flexion contractures
of his fingers. Two months prior to referral he developed a mass in the
right groin which on biopsy gave the diagnosis of amyloidosis. His past
medical history was significant for the onset of arthritis in his shoulders
during World War II, and subsequently in his knees for which he was
discharged from the army. He suffered no articular deformity, however,
and was employed as an iron worker. On physical examination he had a
marked `shoulder-pad' sign (Fig. 1), significant dorsal kyphosis, and
flexion contractures of the fingers. There was loss of touch sensation of
the thumb, index and middle fingers bilaterally with muscle atrophy
consistent with the carpal tunnel syndrome. Elbows were enlarged with
decreased supination of the forearms. His shoulders had very limited
range of motion with practically no external rotation. The wrists showed
soft tissue swelling bilaterally with a decreased range of motion. Hips
had little rotation and there was a soft tissue mass in the right groin.
Evaluation for systemic manifestations of amyloidosis revealed a
creatinine clearance of 39 ml/min with approximately 1.5 g of protein
loss in the urine per 24 h. Technetium 99 pyrophosphate scan showed
uptake in the shoulders, hips, and in the left buttock, corresponding to a
firm mass in that area (Fig. 2). Skeletal survey showed osteopenia, but
no lytic lesions in the skull and no definite erosions in the glenohumoral
joints. The haemoglobin was 8.9 g/dL and bone marrow aspirate from
the right posterior iliac crest give cellular marrow with greater than 20%
plasma cells showing changes consistent with a diagnosis of multiple
myeloma. The rectal biopsy was positive for amyloid, but the skin
biopsy was negative. EMG showed severe median neuropathy at the
wrist. Arthrocenteses of the left knee gave 20 cc of fluid with 300
WBC/mm3. The left shoulder aspirate gave 15 cc of fluid with 350
WBC/mm3. Synovial needle biopsy of the sheath of the long head of the
right biceps tendon gave white granular tissue which on microscopy
showed amyloid infiltration. The echocardiogram was within normal
limits. Immunoelectrophoresis showed a large amount of k light chain
protein in the urine. Prior to discharge from the hospital he had surgical
release of the left carpal tunnel and removal of amyloid-laden material
from the anterior aspect of the left shoulder. This material was frozen at
2 80 8C until used for biochemical analysis. With the diagnosis of
multiple myeloma and amyloidosis the patient was started on
Melphalan and Prednisone, but after receiving three courses at 6
q 2001 Blackwell Science Ltd, Scandinavian Journal of Immunology, 54, 404±408
Shoulder-Pad Sign of Amyloidosis 405
Fig. 1. Patient GRA with typical `shoulder-
pad sign'. Loss of shoulder girdle muscle
mass in systemic amyloidosis accentuates
the soft tissue infiltration by amyloid.
week intervals, he discontinued the therapy. He died in a nursing home
5 months later.
Fibril and protein isolation. Fibrils were isolated from the shoulder
tissue by a modified procedure of Pras et al. [8]. Approximately 3 g of
tissue were homogenized in a blender with 50 ml of 0.1 m sodium
citrate, 0.15 m sodium chloride, and centrifuged for 30 min at
17 000  g The supernatant was discarded and the pellet was
homogenized as above seven more times. The final pellet was
dialyzed against water and lyophilized yielding approximately
210 mg of fibrils. Smears of the material were positive for amyloid
after Congo red staining. Fibrils (100 mg) were suspended in 5 ml of
6 m guanidine hydrochloride, 0.5 m Tris pH 8.5 containing 1 mg
EDTA/ml. The sample was reduced with dithiothreitol (10 mg/ml) at
room temperature for 24 h and alkylated with iodoacetic acid (24.1 mg/
ml). The mixture was centrifuged at 17 000  g for 30 min, and the
supernatant was chromatographed on a Sepharose CL6B column
(2.6  90 cm) equilibrated and eluted with 4 m guanidine
hydrochloride, 0.05 m Tris pH 8.2. Pooled fractions were dialyzed
exhaustively against water and lyophilized.
Gel electrophoresis. Samples were analyzed by SDS-PAGE using the
tricine system of SchaÈgger and Von Jagow [9].
Trypsin digestion and peptide isolation. Protein (2 mg/ml) was
suspended in 0.1 m ammonium bicarbonate, digested with trypsin (2%
by weight) for 18 h at room temperature, and recovered by
lyophilization. The sample was dissolved in 10% acetic acid and the
soluble peptides were fractionated on a Beckman Ultrasphere ODS
column (0.46  25 cm) equilibrated with 0.1% TFA in water and
eluted with an acetonitrile gradient [10]. Separated fractions were dried
in a Savant Speed Vac Concentrator.
Sequence analysis. Samples were analyzed by Edman degradation on
an Applied Biosystems 473 A Protein Sequencer using the
manufacturer's standard cycles.
RESULTS
Chromatography on Sepharose CL6B of reduced and alkylated
fibrils isolated from the shoulder mass yielded a major subunit
406 J. J. Liepnieks et al.
Fig. 2. 99mTechnicium pyrophosphate scan at 3 hours postinjection of
radionuclide shows localization to amyloid infiltrated structures
including the shoulder, hips, and large palpable mass in the left buttock.
protein peak eluting in the approximately 15 kd molecular mass
area with a small shoulder in the approximately 25 kd molecular
mass area. Analysis of the major peak on SDS-PAGE showed
major bands of 11 kd, 14 kd, and 17 kd with a minor band at 25
kd and several very weak bands. The shoulder on the peak
showed a major band at 25 kd and weak bands at 17 kd and 14
kd. Sequence analysis of all four bands after transfer to PVDF
membrane yielded identical major sequence homologous to the
N-terminus of a kappa III Ig light chain. Minor sequences
starting with residue 127 and 150 of kappa constant region were
q 2001 Blackwell Science Ltd, Scandinavian Journal of Immunology, 54, 404±408
also present in the 11 kd band, and with residue 108 and 109 in
the 14 kd band.
The sequence of the variable region of protein GRA was
obtained by direct analysis of the amyloid subunit pool, and by
analysis of peptides obtained from reverse phase HPLC
fractionation of a trypsin digest of the pool (Fig. 3). Peptides
were aligned by homology to kappa light chains. The N-terminal
sequence of GRA identified the protein as belonging to the
kappa III subgroup. Tryptic peptides from residue 109±207 of
the kappa constant region were also present in approximately
half or less the molar amounts of the variable region peptides.
The residue 208±211 tryptic peptide was present in low yields
while the residue 212±214 peptide was not found. The results
suggest that the minor 25 kd band is probably the intact or
nearly intact light chain, and the major lower molecular weight
fragments are the results of proteolysis of the constant region.
Light chain fragments consisting of the variable region or
variable region plus portions of the constant region are the usual
major constituents in AL amyloid deposits with only minor
amounts of intact light chain present.
DISCUSSION
Several features of the variable region sequence of protein GRA
are unique compared to other kappa III light chains [11]. In
CDR1, GRA has His32 and Phe34 instead of the most common
Tyr32 and Ala34. Protein GRA has a two amino acid insert at
residues 95A and 95B. While a few kappa III proteins contain an
additional residue 95A, none have been reported with both
residues 95A and 95B. Protein GRA has Ala59 and Thr95,
residues which are Pro in most kappa III proteins. While the
tertiary structure of a kappa III light chain has not been reported,
the structures of several kappa I light chains have been
determined by X-ray diffraction [12±14]. Assuming that the
general structure of kappa III proteins is similar to kappa I
proteins, residues 59 and 95 would be involved in hairpin turns.
The absence of Pro at these positions and the addition of two
amino acids after residue 95 could alter the conformation of
these turns and affect the b-sheet structures in the b-strands
connected by the turns.
Comparison of protein GRA with three previously character-
ized kappa III amyloid proteins (AL 700, So124, VAG) shows
that, while all have individual unique residues, no feature
common to all four distinguishing them from other kappa III
light chains is apparent [15,16]. None of these amyloid proteins
has an insert between residues 95 and 96 like GRA. Covalently
attached carbohydrate, which is more common in amyloid than
nonamyloid light chains, is present in So124 and VAG but
absent in AL 700 and GRA. Whether any of the unique residues
in these proteins contribute to the amyloidogenicity of kappa III
light chains remains to be determined.
The basis for involvement of the musculoskeletal system,
particularly articular structures, is not known. Based upon the
number of published studies of Ig light chain proteins isolated
from serum, urine, or tissue of patients with amyloid involving

bones or joints, there appears to be a predominance of kappa
light chain proteins [16,17]. This suggests that the primary
structure of the amyloid precursor protein may be an important
factor in the amyloid involvement of the musculoskeletal
system. Kappa III light chain proteins are a rare subgroup of
monoclonal proteins in both multiple myeloma and Ig
amyloidosis. While the structures of kappa III light chain
proteins isolated from amyloid fibrils from the spleen have been
reported [15], the identification of kappa III proteins in amyloid
fibrils from a soft tissue mass [16] and involvement of bone, in
addition to visceral organs [18], are reports similar to the present
case, GRA. Other reports of preferential association of kappa
IIIB light chains with IgM kappa autoantibodies, in particular
with rheumatoid factor activity, suggests an association with
articular disease, but to date no rheumatoid factor activity has
been demonstrated in amyloid patients with the particular kappa
III monoclonal proteins [19]. Another factor based on the few
cases reported is that patients with this form of amyloid
arthropathy which includes the shoulder-pad sign and carpal
tunnel syndrome may be more likely to have multiple myeloma.
In the present case, GRA, a history of inflammatory arthritis was
present, but the role of prior inflammation or injury to articular
structures in the localization of Ig amyloid to these structures can
only be a subject of speculation. To date, no unifying factor has been
identified to explain the rare but unique phenomenon of massive
articular and soft tissue involvement in specific cases of Ig
amyloidosis. As with macroglossia, factors which cause the
`shoulder-pad' sign of amyloidosis remain to be identified.
ACKNOWLEDGMENTS
This work was supported by NIH grants PHS AG 10133,
DK42111, RR-00750, Veteran Affairs Medical Research, the
Marion E. Jacobson Fund, and the Machado Family Research
Fund.
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Shoulder-Pad Sign of Amyloidosis 407
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