Colocalization of Apolipoprotein AI in Various Kinds of Systemic Amyloidosis
Departments of Clinical Neuroscience (NS,MK,MO,MM), Radiopathological Science (TN,YH,TI), and Biomolecular Recognition (HA), Yamaguchi Graduate University School of Medicine, Yamaguchi, Japan
Correspondence to: Naohiro Sakata, MD, Department of Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, 1-1-1, Minami-kogushi, Ube, Yamaguchi, Japan. E-mail: nsakata{at}yamaguchi-u.ac.jp
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
(J Histochem Cytochem 53:237242, 2005)
Key Words: apolipoprotein AI amyloid systemic amyloidosis immunohistochemistry immunoblotting apoAI transgenic mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apolipoprotein AI (apoAI) is a major component of high-density lipoproteins (HDL) (Eisenberg 1984; Coetzee et al. 1986
). It is synthesized in both the liver and small intestine as a 267-amino-acid (aa) prepropeptide. The prepropeptide is cleaved intracellularly to the 249-aa propeptide and secreted into the plasma. Several studies have examined the association between apoAI and amyloidosis (Wisniewski et al. 1995a
,b
; Genschel et al. 1998
). Several forms of amyloidosis, including hereditary systemic amyloidosis (Booth et al. 1996
), hereditary cutaneous and cardiac amyloidosis (Hamidi Asl et al. 1999
), and amyloidosis in atherosclerotic plaques of the human aorta (Westermark et al. 1995
; Mucchiano et al. 2001a
,b
), are caused by apoAI variants. One form of amyloidosis in aortic atherosclerotic plaques is caused by wild-type apoAI (Mucchiano et al. 2001a
). Canine pulmonary vascular amyloidosis is also caused by apoAI (Roertgen et al. 1995
). In addition, it has been reported that apoAI colocalizes with senile plaques in patients with Alzheimer's disease (Wisniewski et al. 1995a
; Harr et al. 1996
).
In the present study we examined colocalization of apoAI in various human systemic amyloidosis immunohistochemically and biochemically. In addition, we examined association of apoAI with murine AA amyloidosis in human apoAI transgenic mice.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunoblotting of Human AL Amyloidosis
For immunoblotting experiments, A amyloid was extracted from the spleen of a patient with primary systemic amyloidosis by the water-extraction method (Pras et al. 1969
; Imada 1981
). A
amyloid deposition was confirmed in a tissue section by Congo red and immunohistochemical staining. The water-extracted material was dissolved in 8 M urea, mixed with Laemmli sample buffer (Bio-Rad; Hercules, CA) containing 5% 2-mercaptoethanol (Sigma; St Louis, MO), and heated at 99C for 6 min. The sample was loaded onto a 1520% gradient gel (Daiichi Pure Chemicals; Tokyo, Japan) for sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore; Billerica, MA). Nonspecific reactions were blocked with 5% skim milk, and the membrane was then incubated with affinity-purified goat anti-human apoAI antibody (1:4000) (Harlan Sera-Lab) for 1 hr at room temperature. Membranes were then incubated with peroxidase-conjugated rabbit anti-goat immunoglobulins (1:1000) (DAKO) for 1 hr at room temperature and visualized with True Blue (KPL; Gaithersburg, MD). As a positive control, purified human apoAI (Biogenesis; England, UK) was resolved on the same SDS-PAGE gel. Immunostaining with anti-
(118134) was also performed to confirm whether the sample contained A
amyloid protein.
To increase confidence in the specificity of the anti-apoAI antibody by examining whether this antibody cross-reacts with other apolipoproteins and amyloid-associated proteins, immunoblotting of apoE (Calbiochem; La Jolla, CA), serum amyloid A (SAA) (American Research Products; Belmont, MA), and serum amyloid P-component (SAP) (Techne; Minneapolis, MN) with affinity-purified goat anti-human apoAI antibody (Harlan Sera-Lab) was also performed.
Immunohistochemical Analysis of ApoAI in Human apoAI Transgenic Mice
ApoAI transgenic mice [C57BL/6-TgN(APOA1)1Rub] (Rubin et al. 1991) were purchased from Jackson Laboratory (Bar Harbor, ME). For the first experiment, AA amyloidosis was induced in the mice according to the method of Ram et al. (1968)
but with some modifications. Briefly, a mixture (0.25 ml) of Freund's complete adjuvant (FCA) and Mycobacterium butyricum was injected subcutaneously into three mice. A second subcutaneous injection of the FCAM. butyricum mixture (0.25 ml) was administered 2 weeks later. These mice were killed under ether anesthesia 2 weeks after the second injection. Spleens were isolated, fixed in 10% formalin, and embedded in paraffin.
For the second experiment, AA amyloidosis was induced in mice by injecting amyloid-enhancing factor (AEF) and AgNO3 to evaluate the earliest stage of amyloid deposition. AEF was prepared from ICR mice according to the method of Axelrad et al. (1982) but with some modifications as described previously (Hoshii et al. 1997
). In three human apoAI transgenic mice, 0.5 ml of AEF was administered intraperitoneally, and 0.5 ml of 2% AgNO3 was administered subcutaneously. Two of these mice were killed under ether anesthesia 72 hr after administration of AEF and AgNO3. Spleens were isolated, fixed in 10% formalin, and embedded in paraffin.
For both experiments, sections (4-µm thick) were cut from the paraffin-embedded tissues, stained with Congo red, and examined by conventional and polarized light microscopy. Immunohistochemical examinations were performed with an indirect immunoperoxidase method as described for human tissues. Rabbit anti-mouse AA (1:1000) (Imada 1981) or affinity-purified goat anti-human apoAI (1:200) (Harlan Sera-Lab) was applied as the primary antibody for 30 min at room temperature. Peroxidase-conjugated goat anti-rabbit immunoglobulin (1:20) (DAKO) or peroxidase-conjugated rabbit anti-goat immunoglobulin (1:50) (DAKO) was applied as the secondary antibody for 30 min at room temperature. Anti-human and anti-mouse apoAI antisera showed <1% cross-species reactivity (Rubin et al. 1991
); therefore, polyclonal anti-human apoAI antibody did not react significantly with murine apoAI. These experiments were carried out under the control of the Guidelines for Animal Experiments of Yamaguchi University School of Medicine.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Involvement of apolipoproteins in amyloidosis has been reported to date. SAA (Benditt et al. 1979; Eriksen and Benditt 1980
) and apoAII (Higuchi et al. 1986
; Benson et al. 2001
) are precursors of major amyloid fibril proteins in humans and mice. ApoE, which has not been known as a major amyloid fibril protein, is a minor constituent of various kinds of systemic amyloidosis (Wisniewski and Frangione 1992
). ApoJ is present in the senile plaques of Alzheimer's disease (Harr et al. 1996
) and may also be a minor constituent of amyloidosis.
ApoAI contains a high proportion of class A amphipathic -helical domains similar to other apolipoproteins, including apoAII, apoJ, and apoE (Hatters and Howlett 2002
). Both full-length apoAI and the N-terminal fragment of apoAI can form amyloid-like fibrils in vitro (Wisniewski et al. 1995a
), and the C-terminal fragment of apoE can also form amyloid-like fibrils in vitro (Wisniewski et al. 1995b
). Furthermore, apoAI and apoE can bind Aß and promote Aß fibrillogenesis in vitro (Castano et al. 1995
; Wisniewski et al. 1995a
,b
). These similarities between apoAI and apoE suggest that the role of apoAI in various kinds of human systemic amyloidosis may be similar to that of apoE. Coincubation of AA with apoE in vitro resulted in a high degree of polymerization of AA peptides, and apoE can also form SDS-resistant AA and Aß complexes (Castano et al. 1995
). These findings suggest that apoE not only promotes amyloidogenesis but also resists proteolysis in vivo. Although similar findings have not been reported for apoAI, it may also promote amyloidogenesis or resist proteolysis in vivo. Chang et al. (2001)
reported that apoE colocalizes with primary and secondary cutaneous amyloidosis but that apoAI is not associated with them. However, the immunostaining method and primary antibody used by Chang et al. (2001)
differed from that used in the present study; therefore, their finding of no apoAI in primary and secondary cutaneous amyloidosis is not surprising.
On the other hand, AA amyloidosis develops in both apoE-deficient mice (Hoshii et al. 1997) and apoAI-deficient mice (Elliott-Bryant and Cathcart 1997
) after inflammatory stimulation. In our immunohistochemical study of AA amyloidosis in human apoAI transgenic mice, we did not observe apoAI immunoreactivity in amyloid deposits at the early stage of murine AA amyloidosis after administration of AEF and AgNO3. Furthermore, in our immunohistochemical analysis of various kinds of human systemic amyloidosis, the immunoreactive area and staining intensity of amyloid deposits were variable within each case. Although it is possible that formalin fixation of tissue specimens may reduce antigenicity and cause uneven staining, apoAI may not be distributed uniformly in amyloid deposits. These findings suggest that apoAI may not be essential for amyloidogenesis.
The exact role of apoAI in amyloidogenesis of various kinds of systemic amyloidosis is unclear. Further studies of the relation of apoAI and amyloid fibril proteins in human systemic amyloidosis are needed. A better understanding of these amyloid-associated proteins may contribute to development of strategies for the treatment of amyloidosis.
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Axelrad MA, Kisilevsky R, Willmer J, Chen SJ, Skinner M (1982) Further characterization of amyloid-enhancing factor. Lab Invest 47:139146[Medline]
Benditt EP, Eriksen N, Hanson RH (1979) Amyloid protein SAA is an apoprotein of mouse plasma high density lipoprotein. Proc Natl Acad Sci USA 76:40924096[Abstract]
Benson MD, Liepnieks JJ, Yazaki M, Yamasita T, Asl K, Guenther B, Kluve-Beckerman B (2001) A new human hereditary amyloidosis: the result of a stop-codon mutation in the apolipoprotein AII gene. Genomics 72:272277[CrossRef][Medline]
Booth DR, Tan SY, Booth SE, Tennent GA, Hutchinson WL, Hsuan JJ, Totty NF, et al. (1996) Hereditary hepatic and systemic amyloidosis caused by a new deletion/insertion mutation in the apolipoprotein AI gene. J Clin Invest 97:27142721
Castano EM, Prelli F, Pras M, Frangione B (1995) Apolipoprotein E carboxyl-terminal fragments are complexed to amyloids A and L. Implication for amyloidogenesis and Alzheimer's disease. J Biol Chem 270:1761017615
Chang YT, Tsai SF, Wang WJ, Hong CJ, Huang CY, Wong CK (2001) A study of apolipoproteins E and A-I in cutaneous amyloids. Br J Dermatol 145:422427[CrossRef][Medline]
Coetzee GA, Strachan AF, van der Westhuyzen DR, Hoppe HC, Jeenah MS, de Beer FC (1986) Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipoprotein composition. J Biol Chem 261:96449651
Eisenberg S (1984) High density lipoprotein metabolism. J Lipid Res 25:10171048[Medline]
Elliott-Bryant R, Cathcart ES (1997) Apolipoprotein E and apolipoprotein A-1 knock-out mice readily develop amyloid A protein amyloidosis. Clin Immunol Immunopathol 85:104108[CrossRef][Medline]
Eriksen N, Benditt EP (1980) Isolation and characterization of the amyloid-related apoprotein (SAA) from human high density lipoprotein. Proc Natl Acad Sci USA 77:68606864[Abstract]
Genschel J, Haas R, Pröpsting MJ, Schmidt HH (1998) Apolipoprotein A-I induced amyloidosis. FEBS Lett 430:145149[CrossRef][Medline]
Hamidi Asl K, Liepnieks JJ, Nakamura M, Parker F, Benson MD (1999) A novel apolipoprotein A-1 variant, Arg173Pro, associated with cardiac and cutaneous amyloidosis. Biochem Biophys Res Commun 257:584588[CrossRef][Medline]
Harr SD, Uint L, Hollister R, Hyman BT, Mendez AJ (1996) Brain expression of apolipoproteins E, J, and A-1 in Alzheimer's disease. J Neurochem 66:24292435[Medline]
Hatters DM, Howlett GJ (2002) The structural basis for amyloid formation by plasma apolipoproteins: a review. Eur Biophys J 31:28[CrossRef][Medline]
Higuchi K, Yonezu T, Kogishi K, Matsumura A, Takeshita S, Higuchi K, Kohno A, et al. (1986) Purification and characterization of a senile amyloid-related antigenic substance (apoSASSAM) from mouse serum. apoSASSAM is an apoA-II apolipoprotein of mouse high density lipoproteins. J Biol Chem 261:1283412840
Hoshii Y, Kawano H, Cui D, Takeda T, Gondo T, Takahashi M, Kogishi K, et al. (1997) Amyloid A protein amyloidosis induced in apolipoprotein-E-deficient mice. Am J Pathol 151:911917[Abstract]
Hoshii Y, Setoguchi M, Iwata T, Ueda J, Cui D, Kawano H, Gondo T, et al. (2001) Useful polyclonal antibodies against synthetic peptides corresponding to immunoglobulin light chain constant region for immunohistochemical detection of immunoglobulin light chain amyloidosis. Pathol Int 51:264270[CrossRef][Medline]
Imada N (1981) Pathological study on amyloidosis: isolation and purification of amyloid fibril protein and biochemical and immunological analysis. Yamaguchi Med J 30:149162
Kitamoto T, Ogomori K, Tateishi J, Prusiner SB (1987) Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids. Lab Invest 57:230236[Medline]
Mucchiano GI, Häggqvist B, Sletten K, Westermark P (2001a) Apolipoprotein A-1-derived amyloid in atherosclerotic plaques of the human aorta. J Pathol 193:270275[CrossRef][Medline]
Mucchiano GI, Jonasson L, Häggqvist B, Einarsson E, Westermark P (2001b) Apolipoprotein A-I-derived amyloid in atherosclerosis. Its association with plasma levels of apolipoprotein A-I and cholesterol. Am J Clin Pathol 115:298303[CrossRef][Medline]
Pras M, Schubert M, Zucker-Franklin D, Rimon A, Franklin EC (1969) The characterization of soluble amyloid prepared in water. J Clin Invest 47:924933
Ram JS, DeLellis RA, Glenner GG (1968) Amyloid 3. A method for rapid induction of amyloidosis in mice. Int Arch Allergy Appl Immunol 34:201204[Medline]
Röcken C, Schwotzer EB, Linke RP, Saeger W (1996) The classification of amyloid deposits in clinicopathological practice. Histopathology 29:325335[Medline]
Roertgen KE, Lund EM, O'Brien TD, Westermark P, Hayden DW, Johnson KH (1995) Apolipoprotein A-1-derived pulmonary vascular amyloid in aged dogs. Am J Pathol 147:13111317[Abstract]
Rubin EM, Ishida BY, Clift SM, Krauss RM (1991) Expression of human apolipoprotein A-I in transgenic mice results in reduced plasma levels of murine apolipoprotein A-I and the appearance of two new high density lipoprotein size subclasses. Proc Natl Acad Sci USA 88:434438
Strege RJ, Saeger W, Linke RP (1998) Diagnosis and immunohistochemical classification of systemic amyloidoses. Report of 43 cases in an unselected autopsy series. Virchows Arch 433:1927[CrossRef][Medline]
Westermark P, Benson MD, Buxbaum JN, Cohen AS, Frangione B, Ikeda S, Masters CL, et al. (2002) Amyloid fibril protein nomenclature 2002. Amyloid 9:197200[Medline]
Westermark P, Mucchiano G, Marthin T, Johnson KH, Sletten K (1995) Apolipoprotein A1-derived amyloid in human aortic atherosclerosis plaques. Am J Pathol 147:11861192[Abstract]
Wisniewski T, Frangione B (1992) Apolipoprotein E: a pathological chaperone protein in patients with cerebral and systemic amyloid. Neurosci Lett 135:235238[CrossRef][Medline]
Wisniewski T, Golabek AA, Kida E, Wisniewski KE, Frangione B (1995a) Conformational mimicry in Alzheimer's disease: role of apolipoproteins in amyloidogenesis. Am J Pathol 147:238244[Abstract]
Wisniewski T, Lalowski M, Golabek A, Vogel T, Frangione B (1995b) Is Alzheimer's disease an apolipoprotein E amyloidosis? Lancet 345:956958[CrossRef][Medline]