The Characteristic Change And Pathological Significance Study Of Immunoglobulin Glycosylation In Multiple Myeloma

Multiple myeloma (MM) is a kind of malignant disease characterized by abnormalproliferation of plasmacyte and a large number of secreted monoclone immunoglobulin (Mprotein) or Bence Jones protein (free monoclone κ or λ chain). The incidence of MM isabout1-2per100,000, mostly occurred in the elder, nearly ten percent of hematologicalcaner and one percent of the whole body malignant tumor. MM could be mostly dividedinto IgG, IgA, light chain, IgD, oligo-secreted or non-secreted and very rarely-occurredIgE. A series of clinical features including osteolytic bone lesions, occasional infection,anemia, hypercalcemia and renal insufficiency are caused by immoderate proliferation andextensive infiltration of malignant plasma cell, which induced the presence and depositionof a number of monoclone immunoglobulin and the inhibition of production in normalimmunoglobulin. The disease still remains incurable and shows multiple clinicalmanifestations with slow onset. The incubation period lasts from several months to morethan ten years accompanied by no symptom, therefore the disease is easily misdiagnosedespecially in early stage. Major challenge is confronted with screen of new biomarkers inearly diagnosis and follow-up monitoring of multiple myeloma. Meanwhile, exploring thenew associated pathogenesis brings light on diagnosis, therapy and prognosis in MM.Abnormal glycosylation is related to metastasis in many cancer cells. As most of theserum N-linked glycoproteins are synthesized by liver and B lymphocyte, the variation ofphysiological function in liver and B lymphocyte could be reflected on changes of serumN-glycans. Changes of glycosylation are hence expected to become the non-invasive earlydiagnostic and differentially diagnostic evidence. The rapid development of several newtechnologies such as DNA sequencer-assisted fluorophore-assisted carbohydrateelectrophoresis (DSA-FACE), HPLC, MALDI-TOF and lectin microarray provide usquick, highly sensitive and high throughout methods to detect N-glycome profile. Thesemethods make serum N-glycosylation tests possible on clinical diagnosis and therapy. Thestudy focuses on the characteristic change, early diagnosis value and related pathogenesisin multiple myeloma relied on the large serum sample of MM, serum purified IgG of IgGMM, pathogenic and regulatory mechanism of abnormal glycosylation in IgG MM celllines.Part1: serum N-glycome and sialic acid analysis in multiple myelomaThis study was based on the analysis of N-glycome in serum glycoproteins to access the charactristic change and clarify the diagnostic and differentially diagnostic value inpartial type of multiple myeloma.Atotal of292MM patients (IgG96, IgA41, light chain42, IgD20and healthycontrol93) and some follow-up patients (including relapse and remission cases) wererecruited to this study. The serum glycoprotein profilings of these samples were detectedand compared according to its type by using DSA-FACE technology. N-glycancharacterization was evaluated by the discrepancy on percentage of N-glycan peaks. TheN-glycans of IgG MM mainly from IgG and depleted IgG was revealed by standardizationof IgG/GB and globulin depleted of IgG/GB. The ROC curve was adopted to access theefficiency of diagnosis and differential diagnosis of specific N-glycan in light chain MM.The results showed as follows: Three N-glycan structure (NGA2F, Peak1; NG1A2F, Peak3and NG1A2F, Peak4) were elevated and two N-glycan structure (NA2, Peak5and NA3,Peak8) were declined in IgG MM. The fluctuation of the N-glycan peaks wereaccompanied by the state of illness (remission and relapse). The abundance of non-galactobiantennary glycans (NGA2F/(IgG/GB)%, Peak1/(IgG/GB)%and NGA2FB/(IgG/GB)%,Peak2/(IgG/GB)%) derived from IgG dropped obviously while the abundance oftriantennary and quadriantennary glycans (NA3/[(GB-IgG)/GB]%, Peak8/[(GB-IgG)/GB]%; NA3Fb/[(GB-IgG)/GB]%, Peak9/[(GB-IgG)/GB]%;NA3Fc/[(GB-IgG)/GB]%, Peak10/[(GB-IgG)/GB]%; NA4/[(GB-IgG)/GB]%, Peak11/[(GB-IgG)/GB]%and NA4Fb/[(GB-IgG)/GB]%, Peak12/[(GB-IgG)/GB]%) originatedfrom depleted IgG rose distinctively in IgG MM. The level of lens culinaris agglutinin(LCA), phytohemagglutinin-erythrocyte type (PHA-E) and maackia amurensisleukoagglutinin (MAL) showed apparently high expression in IgG MM by using serumlectin blot analysis. Mono-galactose biantennary N-glycan (NG1A2F，Peak3) diagnosedlight chain MM with88.1%sensitivity and90.5%specificity for the optimal cut off valueat5.15%. Meanwhile the ROC curve was applied to access the diagnostic effect as the areaunder the curve (AUC) reached0.943. Compared with IgG MM, Peak3differentiallydiagnosed light chain MM with100%sensitivity and78.6%specificity for the optimal cutoff value at6.77%. The ROC curve was applied to access the differentially diagnosticeffect as AUC reached0.895. The N-glycans biomarker Peak3took an advantage onroutine tests such as serum protein electrophoresis and immunofixation electrophoresis forits high sensitivity in light chain MM diagnosis and differential diagnosis. Conclusion:N-glycan profile was altered by the distribution of serum protein in IgG MM. The depleted IgG part is similar to other malignant cancers; the IgG section is characterized by thedecrease of non-galacto biantennary N-glycans. Serum sialic acid was elevatedsignificantly in IgA, light chain and IgD multiple myeloma except IgG multiple myeloma,as compared with healthy control. The ROC curve was applied to evaluate the diagnosticeffect of SA, theAUC reached0.873,0.760and0.845in IgA, light chain and IgD multiplemyeloma.Part2: IgG N-glycomic study in IgG multiple myelomaWe studied on N-glycan profilings of serum purified IgG combined with lectin-blot ofpurified IgG to reveal the characteristic change and potential pathogenic mechanism indifferent disease stage of IgG MM.Afterwards the potential role of key glycosyltransferases was validated in bone marrow.26IgG MM patients and16healthy controlsamples of purified IgG exhibited7main N-glycan peaks by DSA-FACE. Meantime,24IgG MM patients and12healthy control samples of purified IgG processed the relatedlectin blot assay. The research found the non-galacto biantennary glycans (NGA2F, Peak1and NGA2FB, Peak2) apparently decreased in IgG MM group as compared with healthycontrol group, but the bigalacto biantennary glycans (NA2, Peak5; NA2F, Peak6andNA2FB, Peak7) increased significantly and the mono-galacto biantennary glycans had nostatistical variation. We assumed that the expression level of galactose was elevated in IgGMM, The relevant metabolic enzyme---beta1,4-galactosyl transferase polypeptide1(B4GALT1) may play the key role. The purified IgG lectin blot display the higherexpression of sambucus nigra agglutinin (SNA) and the lower expression of concanavalinA(ConA) and phytohemagglutinin-erythrocyte type (PHA-E) in IgG MM group afterstandardizing the IgG stained with coomassie brilliant blue. The expression level ofErythrina cristagalli lectin (ECL)/ConAand SNA/ConAelevated obviously bystandardizing the N-glycans located at IgG heavy chains. Bone marrow samples collectedfrom7IgG MM and3anemia (control) patients was verified to prove the expression levelof B4GALT1, alpha-2,6-sialyltranferase1(ST6GAL1) and relevant lectin ECL, SNA.Allthese enzymes and lectins had the trend of rising. ST6GAL1, SNAand ECL showedstatistical increase in IgG multiple myeloma. Conclusion:Abnormal increase ofgalactosylation and sialylation is associated with pathogenesis in IgG MM. The existenceof abnormal glycosylation, its pathological significance and possible mechanism ispreliminary emerged in IgG MM.Part3: abnormal glycosylation mechanism and functional study in IgG multiple myeloma cell lineIn this part, lentiviral vector was adopted to interfere alpha-2,6-sialyltranferase1(ST6GAL1) and beta1,4-galactosyl transferase polypeptide1(B4GALT1) expression inIgG MM cell line(LP-1). The mRNAand protein expression level of ST6GAL1andB4GALT1was detected by real-time RT-PCR and WB to prove interference efficiency. Todisclose the biological behaviour change in IgG multiple myeloma cell line after RNAsilence, Annexin V/PI double stain cell apoptosis and cell cycle test were used toelucidate the pathogenesis and progression in IgG MM. Results showed the elevation ofearly and late apoptosis ratio in LP-1cells, while the reduction of viable cell ratio and thecell cycle was arrested at G0/G1phase after the interference of ST6GAL1.. Meanwhile, thedown regulation of phosphorylated P38MAPK and NF-κB, accompanied by up regulationof phosphorylated MEK1/2and SAPK/JNK(p46) was observed. The reduction of earlyapoptosis ratio in LP-1cells, while late apoptosis ratio, viable cell ratio and every phase incell cycle had no significant change after the interference of B4GALT1. Therefore,ST6GAL1inhibited cell apoptosis and enhanced cell proliferation. The high level ofST6GAL1might promote the pathogenesis in IgG MM, but the influence of B4GALT1exhibited little on cell apoptosis and cell cycle.In a word, N glycosylation and sialic acid modification had its own character indifferent type MM and could be applied as the biomarkers to early diagnose, differentiallydiagnose, monitor and remedy in multiple myeloma. Abnormal elevation of galactosylationand sialylation was closly related to the pathogenesis in IgG MM, especially thatglycosyltransferase ST6GAL1correlated well with cell cycle, apoptosis andphosphorylated signaling pathway, which might indicate a new potential etiological factorand efficient therapeutic target.