Fragments of the Constant Region of Immunoglobulin Light Chains Are Constituents of AL-Amyloid Proteins Karen Ege Olsen,* ,1 Knut Sletten,† and Per Westermark* *Division of Molecular and Immunological Pathology, Linko ¨ping University, Sweden; and †Department of Biochemistry and Biotechnology Center of Oslo, University of Oslo, Norway Received September 3, 1998 Immunoglobulin light chains are the precursor pro- teins of AL-amyloidosis. In the fibril formation process properties of the variable part of the immunoglobulin light chains are believed to be of major importance. In this work it is shown that fragments of the constant part of the immunoglobulin light chain are a constit- uent of the AL-amyloid proteins of kappa type. A spe- cific antiserum has identified these fragments in gel filtration fractions where the absorbance approached the base line after the main retarded peak. The frag- ments are small and have been overlooked previously in the purification process. The significance of the con- stant part in AL-proteins is unclear, but adds new aspects to the discussion of pre- or post-fibrillogenic cleavage of the immunoglobulin light chains. © 1998 Academic Press Key Words: amyloidosis; kappa; constant region; fibril formation; gel filtration; ELISA. Amyloidoses are protein storage diseases with depos- its of amyloid characterized by fibrillar proteins in -pleated sheets. One of the most common amyloidoses is AL (amyloid light chain) -amyloidosis which can be found both as a local and a systemic form. The precur- sor protein is a monoclonal immunoglobulin light chain which in its native form has a tertiary protein struc- ture rich in -sheets. The pathogenesis of fibril for- mation from immunoglobulin light chains in AL- amyloidosis is unknown, but is probably multifactorial involving properties of the proteins, associated sub- stances and local tissue factors. AL-proteins of -type are more common than -type proteins in contrary to the ratio of : being 2:1 in the related disease multiple myeloma. In addition, the sub- type VI is found exclusively in amyloidogenic light chains, while some other light chain subtypes are more rare as AL-proteins, e.g., only recently we have described the first amyloid protein derived from light chain V subtype (1). These facts indicate that properties of the proteins play an important role for amyloidogenicity. Amino acid sequence analyses have stated that oth- erwise rare amino acid substitutions are frequent in amyloidogenic light chains, although no single amino acid substitution nor any specific pattern of amino acid substitutions have been shown to be responsible for fibril formation (2). Protein analyses have shown that AL-proteins are made of fragments of monoclonal immunoglobulin light chains together with small amounts of intact light chain (3, 4). Amino acid se- quence analyses have revealed that the fragments are derived from the N-terminal part of the variable seg- ment of the immunoglobulin light chain with different sized part of the constant segment. Recently, we have reported a case (VAG) (5) of AL-amyloidosis, where the constant part of immunoglobulin kappa light chain constituted the main part of the AL-protein. This un- usual finding incited the search for the constant part in other AL-proteins. In the present report we show that most AL-proteins of -type contain fragments of the C-terminal part of the constant segment as well as full-length immunoglobulin light chain. MATERIAL AND METHODS In the laboratory archives frozen or lyophilized fibrillar material from 19 cases of AL-amyloidosis were found, representing both local and systemic AL-amyloidosis. The subtype of the cases were known from previous amino acid sequence analysis or from immunoreactiv- ity in ELISA or Western blot with anti-kappa antisera. Amyloid fibril protein purification. After homogenization of tis- sue with amyloid in 0.15 M NaCl, 0.05 M Na-citrate buffer (6), amyloid fibrils were extracted with water as described by Pras (7), with the modification that the pellet material was used as well as the protein fibrils in the supernatants. The amyloid fibril material was lyophilized. In cases of subcutaneous fat biopsies the material was initially put in alkaline ammonium chloride in 0.01 M Tris HCl buffer, pH 8.0, for 2 hours, followed by rinsing in 0.15 M NaCl and finally distilled 1 To whom correspondence should be addressed. Karen Ege Olsen MD, Department of Pathology, Odense University Hospital, DK-5000 Odense C, Denmark. Fax +45 6591 2943. E-mail: karen.ege.olsen@ ouh.dk. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 251, 642– 647 (1998) ARTICLE NO. RC989508 642 0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.