Madden, Benjamin

Abstract AL is a rare plasma cell disorder, resulting from clonal bone marrow plasma cells secreting immunoglobulin light chains (LC), which aggregate abnormally into fibrils, and leads to amyloid deposits in different organs. Previous studies using LC immunopurification and MALDI-TOF/MS (MASS-FIX) on patients with known plasma cell disorders (PCDs), demonstrated that monoclonal LC from AL patients display unusually complex mass to charge spectra (m/z) for their LCs at much higher frequencies than other PCDs such as multiple myeloma. We hypothesized that these were post-translational modification, presumably glycoforms. Here, we report investigations into LC glycosylation using MASS- FIX. Serum from 212 lambda and 72 kappa AL patients were collected following Mayo Clinic IRB guidelines. LC was purified from serum using nanobody enrichment method, followed by MASS-FIX. The LCs spectra acquired on MASS-FIX were visually analyzed and were classified into different patterns. The kappa and lambda LC mass distributions with abnormal complex patterns were interrogated for the presence of glycosylation by exposure to the LC to a deglycoslylating enzyme (PNGase F) and analysis of LC mass distribution using MASS-FIX and by LC-ESI-Orbitrap. Monoclonal LCs demonstrating significantly mass shifted peak outside the normal kappa and lambda mass distribution were observed in 31% of kappa and 6% of lambda patients with AL. In order to determine if this characteristic mass shift was due to glycosylation, 16 kappa and 6 lambda LCs were treated with a PNGase F and LC spectrum were re-examined by MASS-FIX and LC-ESI-Orbitrap. The monoclonal LCs whose masses were significantly shifted beyond normal kappa and lambda mass distributions were found to shift to lower molecular weight after PNGase F treatment, confirming the presence of N-glycosylation. Moreover, an additional 5 kappa and 23 lambda samples had other complex patterns, typically with multiple additional peaks at increments of 162 Da. These samples did not shift with PNGase F suggesting either glycation or O-glycosylation. This work is significant as it confirms that N-glycosylated LCs are over represented in patients with AL in comparison to other PCDs. In patients with AL, the frequency of N-glycosylation of kappa LC clones was 5 times higher than that of lambda LC clones. More work is needed to explore the mechanism for this finding, the role of glycosylation in fibril formation, and the other complex patterns observed. In addition, MASS-FIX method used in this work is currently being validated in the clinical lab such that rapid detection of N-glycosylation in patients with monoclonal gammopathies will be possible routinely, potentially leading to earlier clinical suspicion for AL among patients with monoclonal gammopathies and hence to earlier detection of AL. Citation Format: Sanjay Kumar, Angela Dispenzieri, Surendra Dasari, Paolo Milani, Giampaolo Merlini, MeLea Hetrick, David Barnidge, Benjamin Madden, David Murray. Mass spectrometric approach to identify N-glycosylation of light chain in patients with immunoglobulin light chain amyloidosis (AL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2693.

Two putative ribonucleases have been isolated from the secondary granules of mouse eosinophils. Degenerate oligonucleotide primers inferred from peptide sequence data were used in reverse transcriptase-PCR reactions of bone marrow-derived cDNA. The resulting PCR product was used to screen a C57BL/6J bone marrow cDNA library, and comparisons of representative clones showed that these genes and encoded proteins are highly homologous (96% identity at the nucleotide level; 92/94% identical/similar at the amino acid level). The mouse proteins are only weakly homologous (approximately 50% amino acid identity) with the human eosinophil-associated ribonucleases (i.e., eosinophil-derived neurotoxin and eosinophil cationic protein) and show no sequence bias toward either human protein. Phylogenetic analyses established that the human and mouse loci shared an ancestral gene, but that independent duplication events have occurred since the divergence of primates and rodents. The duplication event generating the mouse genes was estimated to have occurred < 5 x 10(6) years ago (versus 30 to 40 x 10(6) years ago in primates). The identification of independent duplication events in two extant mammalian orders suggests a selective advantage to having multiple eosinophil granule ribonucleases. Southern blot analyses in the mouse demonstrated the existence of three additional highly homologous genes (i.e., five genes total) as well as several more divergent family members. The potential significance of this observation is the implication of a larger gene subfamily in primates (i.e., humans).