BIOCHIMICA ET BIOPHYSICA ACTA BBA 35838 N-TERMINAL AMINO ACID ANALYSIS OF THE AMYLOID FIBRIL PROTEIN* 183 MARTHA SKINNER AND ALAN S. COHEN The Arthritis and Connective Tissue Disease Section of the Evans Department of Clinical Research, University Hospital, and the Department of ,14edicine, Boston University School of :~ledicine, Bosto~z University Medical Center, Boston, Mass. o2zz8 (U.S.A.) (Received December I5th, 197 o) SUMMARY I. The N-terminal amino acid of the amyloid fibril protein was studied by the method of Sanger using paper chromatography and high voltage electrophoresis. The amyloid fibrils were obtained from splenic homogenates of 14 patients with primary, secondary, or myeloma-associated amyloidosis. Aspartic acid was the amino-terminus in two preparations from primary amyloidosis and glutamic acid in one primary, one secondary and one myeloma associated amyloid. Nine amyloid fibril preparations (five from primary and four from secondary amyloidosis) did not have a detectable amino-terminus by this method. One of these nine preparations was further investigated and found to have a pyroglutamic acid amino-terminus. A small amount of N-terminal serine was found in all 14 preparations. 2. This result is the first evidence of a chemical difference in the amyloid fibril proteins from different sources. The variations in the N-terminal amino acids did not correlate with the clinical types of amyloidosis. INTRODUCTION Amyloidosis is a disorder of unknown etiology in which a fibrous protein is laid down in the extracellular connective tissue of various organs of the body. The clinical types include 1-3 primary amyloid, that with no antecedent or coexisting di- sease; secondary, amyloid associated with a chronic inflammatory or infectious disease; and amyloidosis associated with multiple myeloma or other tumors. The chemical and physical properties of the amyloid fibril protein have been under increasing investigation as it has been isolated from the connective tissue in recent years. In the electron microscope the amyloid fibril appears as a non-branching fibrous structure consisting of a number of filaments aggregated side by sidO. X-ray ~-A portion of this work was presented in abstract on June 19, 1969 at the meeting of the American Rheumatism Association (Arthritis Rheumat., 12 (1969) 333). Biochim. Biophys. Acta, 236 (1971) 183--190
Transcript
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35838
N-TERMINAL AMINO ACID ANALYSIS OF THE AMYLOID F I B R I L
PROTEIN*
183
MARTHA SKINNER AND ALAN S. COHEN The Arthritis and Connective Tissue Disease Section of the Evans Department of Clinical Research, University Hospital, and the Department of ,14edicine, Boston University School of :~ledicine, Bosto~z University Medical Center, Boston, Mass. o2zz8 (U.S.A.)
(Received December I5th, 197 o)
SUMMARY
I. The N-terminal amino acid of the amyloid fibril protein was studied by the method of Sanger using paper chromatography and high voltage electrophoresis. The amyloid fibrils were obtained from splenic homogenates of 14 patients with primary, secondary, or myeloma-associated amyloidosis. Aspartic acid was the amino-terminus in two preparations from primary amyloidosis and glutamic acid in one primary, one secondary and one myeloma associated amyloid. Nine amyloid fibril preparations (five from primary and four from secondary amyloidosis) did not have a detectable amino-terminus by this method. One of these nine preparations was further investigated and found to have a pyroglutamic acid amino-terminus. A small amount of N-terminal serine was found in all 14 preparations.
2. This result is the first evidence of a chemical difference in the amyloid fibril proteins from different sources. The variations in the N-terminal amino acids did not correlate with the clinical types of amyloidosis.
INTRODUCTION
Amyloidosis is a disorder of unknown etiology in which a fibrous protein is laid down in the extracellular connective tissue of various organs of the body. The clinical types include 1-3 primary amyloid, that with no antecedent or coexisting di- sease; secondary, amyloid associated with a chronic inflammatory or infectious disease; and amyloidosis associated with multiple myeloma or other tumors.
The chemical and physical properties of the amyloid fibril protein have been under increasing investigation as it has been isolated from the connective tissue in recent years. In the electron microscope the amyloid fibril appears as a non-branching fibrous structure consisting of a number of filaments aggregated side by sidO. X-ray
~-A portion of this work was presented in abstract on June 19, 1969 at the meeting of the American Rheumatism Association (Arthritis Rheumat., 12 (1969) 333).
Biochim. Biophys. Acta, 236 (1971) 183--190
184 M. SKINNER, A. S. COHEN
diffraction has shown that it has a cross-fl configuration 5. The mucopolysaccharide content is o.4°/o 6 and the total carbohydrate is 5°b 7 . . Amino acid analyses indicate a high content of acidic amino acids with aspartic acid and glutamic acid amounting to 2o% of the totaF, 8. There are no uniquely absent amino acids except those found in collagen (hydroxyproline and hydroxylysine) and those found in elastin (desmosine and isodesmosine). These characteristics, i.e. ultrastructural appearance, X-ray diffraction pattern, carbohydrate and amino acid analyses, have been identical in the amyloid fibrils from the various clinical types of amyloidosis studied.
Another protein component of amyloid, P-component, has been isolated from amyloidotic tissue and has been shown to be immunologically identical with a circulating alpha globulin. P-component, a pentagonal rod, differs from the fibril in its ultrastructure, chemistry and immunologic interactions and constitutes only a minute fraction of amvloid deposits "-u.
This paper reports a study of the N-terminal amino acids of the major com- ponent, the amyloid fibril protein. Tissue from patients with the various types of amyloidosis were used as the sources of the fibril in order to assess any similarities or differences that might exist in the N-terminal residues amongst these types.
METHODS
Preparation of material Fourteen amyloid fibril preparations were obtained from human autopsy
spleen tissue by a method similar to those reported 7,12. 20 g of amyloid-laden spleen was homogenized with 20o ml saline in a ground-glass hand homogenizer and centri- fuged at 15 ooo rev./min (27 ooo x g) at 4 ° for 3o rain. The sediment was repeatedly homogenized with additional saline until the absorbanee of the supernatant was o.075 at 280 nm in a Zeiss spectrophotometer PMQ II. The top layer of the sediment was collected, then further homogenized with water and centrifuged at I8 ooo rev./ rain (39 IOO x g) for I h. This was repeated three times with the final centrifugation at 12 ooo rev./min (18 800 x g). Amyloid fibrils were obtained by lyophilization from suspension in the two final supernatants and from the top layer of the sediment.
The insoluble nature of the amyloid fibril has been noted to cause difficulty in obtaining a pure preparation. Tile protein prepared by tile above method proved to be relatively pure when assessed by the %llowing criteria.
(I) All preparations were examined by electron microscopy and had the characteristic fibrils previously described 4. No other structural proteins, such as collagen or P-component, were identifiable.
(2) All exhibited the well-defined green birefringence in the polarizing micro- scope after Congo red staining la indicating that this indeed was amyloid.
(3) Amino acid analyses were consistent among the various preparations showing the high content of acidic amino acids 7,8.
(4) X-ray diffraction patterns showed a cross-/~ configuration a. (5) Extracts from the fibrils did not contain IgG or P-component when tested
against the specific antiserum by agar diffusion using Ouchterlony plates. To de- termine whether or not immunoglobulins might be more tightly bound to the fibril and not identified by this technique, fluorescence microscopy was done. Embedded
Biochim. Biophys. Acta, 236 (1971 ) 183-19o
N-TERMINAL ANALYSIS OF AMYLOID FIBRIL PROTEIN I8 5
amyloid fibrils were sectioned 6 #m thick and reacted with fluorescinated anti IgG. By this method IgG was demonstrable in 2 of 3 amyloid fibril preparations tested.
N-terminal analysis The amino-terminus of the protein was labeled with FDNB according to the
method of SANGER 14 as modified by LEVY la. Approx. 15 mg of the amyloid fibril protein and an equal quanti ty of NaHCOa in 5 ml water were reacted with 5 ml of an ethanolic 5% FDNB solution by shaking in the dark at room temperature for 2 tl. Tlle labeled protein was hydrolyzed under nitrogen in 5-7 M HC1 at IiO ° for 16 11. To test for the presence of a more acid labile N-terminal glycine or proline, another portion of the labeled protein was hydrolyzed in concentrated HC1 for 4 h. The 2,4- dinitrophenyl-labeled (DNP-labeled) amino acids were extracted from the hydrolysate with ether, and excess dinitrophenol was removed by sublimation.
A two dimensional system of paper chromatography was used to separate the ether-soluble DNP-labeled amino acids 15. A toluene 2-chloroethanol pyridine (3o:18:9, by vol.) aqueous ammonia solvent was used in the first (ascending) di- mension and 1.5 M phosphate buffer (pH 6) was used in the second (descending) dimension. The separated yellow spots were compared with a standard DNP-labeled amino acid map, eluted in a I °/o NaHCO 3 solution and quantitated. Identi ty of tile DNP-labeled amino acid was confirmed by high voltage electrophoresis on a Savant flat plate 30 A using pyridine-acetic acid water (I :1o:39 o, by vol.) at pH 3.5.
Because IgG had been found in two of the fibril preparations by fluorescence microscopy, N-terminal analysis of 15 nag of partially purified Fraction I I IgG was performed by the above procedure in order to compare the results with those for amyloid fibrils under the same conditions.
Further studies (a) To test for the presence of asparagine or glutamine, which would be con-
verted to the corresponding dicarboxylic acid by acid hydrolysis, an alternate method of hydrolysis using leucine amino peptidase (EC 3.4.1.1) was carried out 16. 2o-3o nag of the FDNB-labeled amyloid was hydrolyzed with I mg leucine amino peptidase for 4-20 h at 4 o°.
(b) To confirm the N-terminal amino acid the DNP-labeled amino acid was transformed into its unsubstituted form by hydrolysis at IOO ° for 2 h with 0.2 ml concentrated ammonialL This was followed by ascending paper chromatography in n-butanol-acetic acid-water (4:1:5, by vol.) and visualization of the amino acid after spraying with 0.29/0 ninhydrin.
(c) To test for the presence of arginine, the water-soluble DNP-labeled amino acids remaining in the hydrolysates after ether extraction were concentrated by rotary evaporation at 37 ° and examined by high voltage electrophoresis. Whatman No. 3 paper was used in a solvent system of 0.6 M formic acid and 2.1 M acetic acid ( I : i , by vol.), pH 1.85. The paper was sprayed with a modified Sakaguchi reagent to identify arginine 18.
(d) To test for the presence of cysteine, performic acid oxidation of the protein 19 was carried out followed by the reaction with FDNB.
(e) To test for the presence of pyroglutamie acid as an N-terminal, the fibrils of one preparation which did not yield an amino-terminus with FDNB were digested
Biochim. Biophys. dcta, 236 (1971) 183-19o
186 M. SKINNER, A. S. COHEN
with pronase at 60 ° for 24 h. This particular N-terminal peptide thus could be sepa- rated from the other peptides by elution with water on a Dowex 5o-2X (H +) column since the peptides with free a-amino groups were retained on the column 2°. An amino acid analysis of this N-terminal peptide showed the following amino acids in decreas- ing amounts: glutamic acid, serine, aspartic acid and alanine. The peptide was treated with I M NaOH for 2o min at IOO ° to convert a possibly pyroglutamyl peptide to a glutamyl peptide, neutralized with I M HC1 to pH 8.o, then subjected to the previously described N-terminal analysis, e0
o..| NH,
Toluene
I
-- Q Trp
AIo O IbVoe LeU ~ DNP OH
tl~ Set
l ~ Glu or Alp
Fig. I. Paper chromatograph of the amyloid and s tandard DNP-Iabeled amino acids. The DNP- labeled amino acids from an amyloid fibril protein are seen in a drawing of a typical chromato- graph (right) and compared to s tandard DNPdabeled amino acids (left). Note the proximi ty of aspart ic and glutamic acids. A toluene buffer system was used in the first dimension and t. 5 M phospha te buffer in the second dimension.
RESULTS
Tile dinitrophylated N-terminal of 5 of the 14 amyloid fibril preparations resolved in the aspartic-glutamic region of the chromatograph (Fig. I). In 9 of the 14 preparations there was no significant amount of any DNP-labeled amino acid. In all 14 amyloid fibril preparations, the ether-soluble DNP-labeled amino acids from the 4-11 hydrolysate did not yield any DNP-glycine or proline. The water phase of these hydrolysates contained only e-DNP-lysine and no arginine. Performic acid oxidized protein did not reveal any cysteine.
The dinitrophenylated N-terminal of the 5 preparations which resolved in the aspartic-glutamic acid region of the chromatograph was identified by high voltage electrophoresis as aspartic acid in two amyloid fibril preparations (both from patients with pr imary amyloidosis) and glutamic acid in the three other amyloid fibril prepa- rations (from one patient with primary, one with secondary, and one with myeloma associated amyloidosis) (Fig. 2). In addition to aspartic acid or glutamic acid a small amount of DNP-serine was consistently present on the paper chromatograms from all the amyloid fibril preparations. The relative amounts of aspartic acid and/or glutamic acid and serine in these five amyloid fibril preparations is listed in Table I.
Biochim. Biophys. Acta, 236 (1971) 183-19o
N-TERMINAL ANALYSIS OF AMYLOID FIBRIL PROTEIN 187
A S P - G L U
S a m p l e A
G L U
A S P
S a m p l e B
A S P - G L U
Fig. 2. High voltage electrophoresis of the amyloid fibril protein amino-terminus. High voltage electrophoresis of the spot eluted from the paper chromatograph clearly indicated aspartic acid as the amino-terminus of Sample A and glutamic acid as the amino-terminus of Sample B when compared to DNP-standards.
The N- terminal amino acid analyses carried out on the amyloid fibrils of the 9 addi t ional pat ients (5 pat ients with pr imary and 4 with secondary amyloidosis) which did not yield a significant amoun t of any amino acid, did show trace amounts of aspartie acid or glutamic acid (Table II). Trea tmen t of the N- terminal peptide of one of these preparat ions (66 63) with I M NaOH followed by N-terminal analysis resulted in the identification of glutamic acid. Therefore, in this preparat ion pyro- glutamic acid is the most likely amino- terminus . Perhaps it is also the amino- terminus of the other preparat ions without identifiable N-terminals whose N-terminal peptide was not similarly analyzed. DNP-serine was found in approximately the same q u a n t i t y in these amyloid fibril proteins as in the previous group.
TABLE [
N-TERMINAL AMINO ACIDS OF AMYLOID FIBRIL PROTEIN Corrections were not made for hydrolytic or chromatographic losses.
Type of Patient amyloidosis No.
Quantity of amino acid (moles/Ioo ooo g)
Asp Glu Set
Primary 65-15 o.74o o. i o 3 o. I o8 68-43 o.444 trace o.o66 66-1o 0.020 0.200 0.040
Myeloma 65-62 Trace o.63o 0.093
Secondary 64-67 0.052 0.238 0.079
IgG o.171 0.248 - -
Biochim. Biophys. Acta, 236 (1971) 183-19o
1 8 8 M. SKINNER, A. S. COHEN
TA BLE 11
N - T E R M I N A L A M I N O ACIDS OF A M Y L O I D F I B R I L P R O T E I N
Type of Patient Quantity of amino acid (moles/ioo ooo g) amyloidos~s No. -
The results of N-terminal analysis of IgG performed by the same Sanger-Levy procedure are compared to those for amyloid fibrils in Table I. In two amyloid preparations, 65-15 and 68 43, the amount of aspartic acid is significantly higher than the amount for an equal quantity of IgG. The amount of glutamic acid in the amyloid fibrils is considerably less than that of IgG and the ratio of aspartic acid to glutamic acid is reversed. In the other three preparations, 66 IO, 65 62 and 6 4 67,
the amount of glutamic acid is higher than that of IgG in one, 65-62, and the amount of aspartic acid is much less in all. In no case was the molar ratio of aspartic acid to glutamic acid similar to that of IgG.
D I S C U S S I O N
We previously reported glutamic acid as the amyloid fibril protein amino- terminus 21. As more preparations have been studied aspartic acid has also been identified. The finding of two different N-terminal amino acids in different amyloid fibril proteins as well as an unreactive amino-terminus is the first evidence of chemical differences in the amyloid fibril. Although differences might be expected in the various clinical types of amyloidosis, to date the ultrastructure, staining properties, amino acid analyses and ultracentrifugal analyses of the fibrils extracted from these various types of amyloid have all been comparable. The different N-terminals in this study were not clearly related to the type of amyloid. There were 8 primary amyloid fibril preparations of which two had aspartic acid, one glutamic acid, one pyroglutamic acid and 4 unidentified N-terminals. Only I myeloma amyloid was tested and that had a glutamic acid N-terminal, while of the 5 secondary amyloids tested, 4 had no identifiable N-terminal and one had glutamic acid.
The presence of a small amount of contaminating IgG was demonstrated by fluorescence inicroscopy in some preparations. Aspartic acid and glutamic acid are both known to be N-terminals of immunoglobulins 22-24. However, when comparing the quantitative results of the N-terminals of the amyloid fibrils with those of IgG (Table I), the large amount of DNP-aspartic acid found in preparations 65 15 and 68-43 with small amounts of DNP-glutamic acid strongly suggests that aspartic acid is the N-terminal of these amyloid fibrils. Likewise the large amount of DNP-glutamic
Biochim. Biophys. Acta, 236 (1971) t83-I0O
N-TERMINAL ANALYSIS OF AMYLOID FIBRIL PROTEIN 18 9
acid found in 66-1o, 65-62, and 64-67 with very small amounts of DNP-aspartie acid suggests glutamic acid is the N-terminal of these three fibril preparations when the ratios are compared to the results for IgG. The small amounts of DNP-labeled amino acids in the nine preparations with unidentified N-terminals may be due to only trace amounts of these N-terminals available. The possibility of contaminating IgG contributing to some of these results cannot be excluded.
A small amount of serine was available for DNP-labeling in all of the prepa- rations. This observation deserves further investigation. I t is possibly the amino- terminus of a chain of the amyloid fibril protein which is being partially destroyed during the procedure. Another possibility is that it is a link to the carbohydrate fraction of the protein.
In summary, these chemical studies have indicated a heterogeneity of the amyloid fibril protein. Amino-terminal differences are not uncommon in the same protein which has been obtained from various sources. For example, the amino- terminal amino acid of immunoglobulin light chains has been reported to be aspartie acid 22, serine 2~, or pyrrolidone carboxylie acid 24. I t is interesting to note that recent immunologic studies on the amyloid fibril have also demonstrated heterogeneity by non-identity of antiserums to antigenic fragments from different preparations of the fibrils~5, 26.
ACKNOWLEDGMENTS
The authors wish to express their thanks to Dr. K. Schmid and Dr. M. Ishiguro, Boston University Medical Center, for invaluable advice and for the use of the high voltage eleetrophoresis equipment, and to Marcia Lacala and Cynthia Cordas for expert technical assistance. Grants in support of these investigations were received from the U.S. Public Health Service, National Insti tute of Arthritis and Metabolic Diseases, Grant Nos. AM-o4599 and TI-AM-5285, from the Massachusetts Chapter of the Arthritis Foundation, and from the Arthritis Foundation.
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