L. A. Omtvedt

Glycosylation of immunoglobulin light chains associated with amyloidosis

AL amyloidosis is a fatal disease caused by deposition of immunoglobulin light chains in a fibrillar form (AL) in various organs. By searching the Kabat database of immunoglobulin sequences using the KabatMan software, we have shown that there is a preponderance of the consensus glycosylation sequon (AsnXxxSer/Thr) in the framework regions of amyloid light chains. We have characterised by computer graphics simulations, NMR spectroscopy and carbohydrate biochemistry the structure and conformation of the oligosaccharide from amyloid protein AL MS (λI) and from the amyloid associated Bence Jones protein of patient MH (K1). These proteins have glycosylation in the hypervariable complementarity–determining region versus framework region, respectively. Both contained a 2–6 sialylated core fucosylated biantennary chain mostly with bisecting GlcNAc. Together our results suggest that light chain glycosylation may be one of several modifications which may render the protein more prone to amyloid formation.

The Amino Acid Sequence of the Glycosylated Amyloid Immunoglobulin Light Chain Protein AL MS

The authors report on the amino acid sequence of the glycosylated amyloid protein AL MS. The amyloid fibrils were extracted from the spleen of a patient (MS.) with amyloidosis. The protein AL MS was purified from the amyloid fibrils by gel filtration. SDS–PAGE performed on the purified protein material showed glycosylated protein bands in the range of 22 to 32 kDa, corresponding to polymerization of N‐terminal fragments. The protein was characterized by amino acid analysis and Edman degradation. Tryptic digest combined with Staphylococcal V8 protease, chymotrypsin and pyroglutamate aminopeptidase digestion, as well as cleavage with BNPS‐skatole, established the complete amino acid sequence of 168 residues. The protein was compared to other proteins in the SWISSPROT databank, showing homology with the immunoglobulin light chain variable subgroup λI. AL MS showed some unique amino acid substitutions. Highly conserved residues Gly(57) and Arg(61), were exchanged to arginine and glutamine, respectively, possibly altering the three‐ dimensional structure of the protein.

Kinetics of Glycosaminoglycan Deposition in Splenic AA Amyloidosis Induced in Mink

The kinetics of splenic glycosaminoglycan (GAG) expression in mink has been investigated during the course of AA amyloid induction, i.e. at 3 to 6 weeks of lipopolysaccharide (LPS) treatment. Splenic amyloid was demonstrated by means of Congo red staining in five of 19 LPS‐treated mink. Chondroitin/dermatan sulfate (CS/DS), as well as heparan sulfate proteoglycans (HSPG), was extracted from amyloid and control spleens. Independently of the presence of amyloid, the total amount of splenic GAGs increased with the duration of LPS treatment, and an HSPG population was found confined to the LPS‐treated spleens. The differential expression of various PG and GAG epitopes in mink spleen was investigated with the help of immunohistochemistry. The amyloid deposits were shown to contain GAG chains of CS and HS, and the core proteins of DSPG decorin and the HSPGs perlecan and agrin. Decorin and perlecan were shown in normal spleens localized to the splenic ellipsoids, an early target for AA amyloid deposition. The constitutive expression of PGs at predilection sites for amyloid deposition and their increased expression in the tissues developing amyloidosis at these early stages show that PGs are available for the formation and deposition of AA amyloid.

Characterization of Proteoglycans and Glycosaminoglycans in Splenic AA Amyloid Induced in Mink

Amyloidosis of the protein AA type is readily induced in mink using repeated injections of bacterial lipopolysaccharide (LPS). We have characterized splenic proteoglycans/glycosaminoglycans (PGs/GAGs) in mink during amyloidogenesis. Moderate to rich amounts of amyloid exhibiting green birefringence was demonstrated by polarization microscopy of the splenic section stained with Congo red in seven out of eight minks after 10 weeks of LPS‐treatment, and a significant increase in the total amount of PGs and GAGs in AA amyloid spleens was observed (two to eight times that in unstimulated animals). Intact PGs as well as free GAGs were extracted, and heparan sulfate (HS) was the most abundant GAG in the amyloid as well as in the control spleens. The GAGs showing the most pronounced increase in the amyloid spleens was of the chondroitin sulfate/dermatan sulfate (CS/DS) type and these were extracted in the form of free GAG chains. We conclude that there is a selective enrichment of PGs/GAGs in extracted splenic amyloid in the mink, which confirms to previous observations in human amyloid as well as in other animal species, supporting their pathogenic significance in the formation of AA amyloid.

Serum amyloid P component in mink, a non-glycosylated protein with affinity for phosphorylethanolamine and phosphorylcholine.

Experimental AA amyloidosis in the mink is used as a model for the amyloid disease process. In that context it is important to characterize the different proteins involved in the amyloid formation. In the present work, we have characterized the serum amyloid P component (SAP) in mink. SAP was purified from serum by affinity chromatography using phosphorylethanolamine-coupled ECH-sepharose 4B. SDS – PAGE showed one major protein band (∼26 kDa) together with one minor band (10% of the major band) with a higher molecular mass (∼30 kDa) corresponding to a non-glycosylated and a glycosylated variant. All SAP molecules elucidated so far have at least one major subunit that is heavily glycosylated. It is therefore the first time that a non-glycosylated SAP protein is found in a mammalian species. The amino acid sequence was established using Edman degradation and mass spectrometry. As expected, the protein showed high homology with the other mammalian SAP molecules, ranging from 73% (human) to 63% (mouse). The SAP protein showed affinity for phosphorylcholine and thus expressed CRP-like properties.

Serglycin expression in CD2+ and CD14+ cells from patients with various rheumatic diseases

The objective of the study was to look at the in vivo expression of serglycin in cells taken from patients with an inflammatory disease. The mRNA expression of the small proteoglycan serglycin was investigated in macrophages/monocytes and T-cells derived from the synovial fluid and blood of six patients with various rheumatic diseases and from the blood of two control subjects. Our results demonstrate higher levels of expression in CD14+ cells taken from patients with chronic inflammatory diseases than in control subjects. This suggests that serglycin may play a role during the inflammatory process.The objective of the study was to look at the in vivo expression of serglycin in cells taken from patients with an inflammatory disease. The mRNA expression of the small proteoglycan serglycin was investigated in macrophages/monocytes and T-cells derived from the synovial fluid and blood of six patients with various rheumatic diseases and from the blood of two control subjects. Our results demonstrate higher Levels of expression in CD14+ cetts taken from patients with chronic inflammatory diseases than in control subjects. This suggests that serglycin may play a role during the inflammatory process.

The carbohydrate structure of the amyloid immunoglobulin light chain protein EPS

The glycosylated amyloid light chain protein EPS was isolated from amyloid fibrils from the liver of a patient with Waldenstroms macroglobulinemia. A tryptic glycopeptide of EPS (200 nmol) was isolated and characterized by 1H-n. m. r. The glycopeptide was digested with N-glycanase and the released oligosaccharides analyzed by high performance anion exchange chromatography. The monosaccharide composition and the linkage of the different oligosaccharides were determined by methanolysis and methylation analysis. The NMR spectrum revealed a bisected biantennary oligosaccharide as the main structure. HPAEC-PAD demonstrated the presence of several other related glycans in the glycopeptide preparation.