Introduction: Alpha-2-macroglobulin (α2m) is a plasma glycoprotein serine protease inhibitor. Previous studies have shown that coagulation factor concentrations are highly variable with age and α2m levels have been found to be twice as high in newborns compared to adults. This may contribute to a resistance towards thrombotic events observed in young populations. Protein glycosylation is known to affect protein activity and the glycosylation profile of adult α2m has previously been analyzed. Information regarding glycosylation of α2m in other age groups has yet to be elucidated. Therefore, the purpose of this study is to examine the differences in the glycosylation profiles between newborn and adult α2m. Methods: To evaluate glycan macroheterogeneity, plasma samples were enzymatically deglycosylated by PNGaseF, followed by SDS PAGE and western blotting (WB) to detect α2m. To evaluate microheterogeneity, plasma samples were incubated with Neuraminidase (Clostridium perfringens) followed by native PAGE and WB to determine sialic acid content. To detect non-sialylated terminal galactose residues, plasma samples were incubated with immobilized RCA120, and lectin-bound molecules were separated from unbound molecules. Additionally, the affinity of α2m for ricin was evaluated by eluting bound proteins with increasing concentrations of galactose. All fractions were subjected to SDS-PAGE and WB to detect α2m. 2D gel electrophoresis was completed to examine differences in pI and molecular weight of α2m in both age groups. Purification by immunoprecipitation was also performed and eluted α2m was analyzed by fluorescence-assisted carbohydrate electrophoresis (FACE) to determine the glycan fingerprint in the two populations. Results: Deglycosylation of both newborn and adult α2m with PNGaseF resulted in a change in migration and apparent molecular weight, however no statistically significant difference was found between newborn and adult. On native PAGE following treatment with neuraminidase, newborn α2m exhibited a statistically significant change in migration compared to adult. Additionally, newborn α2m exhibited a higher percentage of molecules bound to RCA120 than adult (no statistical difference) and elution of α2m from RCA120 with a galactose step gradient produced similar profiles for newborn and adult molecules. 2D electrophoresis and WB revealed a difference in pI of α2m in newborns as compared to adults. Finally, purified newborn and adult α2m were analyzed by FACE and quantification of prominent fluorescent bands revealed a higher secondary:primary band ratio in newborns when compared to adults. Conclusions: To our knowledge, this is the first study investigating glycosylation differences between newborn and adult α2m molecules. The results from PNGaseF analyses indicate no significant difference in total N-glycan content. Neuraminidase results suggest significantly greater sialic acid presence on newborn α2m, however there was no significant difference in galactose content. 2D electrophoresis revealed a difference in pI as well as the way in which newborn and adult α2m degrade when exposed to experimental conditions. A2m was successfully purified from both newborn and adult plasma, and FACE results indicate that the proportion of more branched glycans present in the two major fluorescent bands are of higher quantity in newborns than adults. / Thesis / Master of Science (MSc)

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No description available.

CD109 is a 170kD glycosylphosphatidylinositol-anchored protein expressed on subsets of fetal and adult CD34+ haematopoietic stem cells, endothelial cells, activated T cells, and activated platelets. Cloning of the CD109 cDNA by our group identified the molecule as a novel member of the alpha2M/C3/C4/C5 family of thioester containing proteins. Curiously, CD109 bears features of both the alpha2M and complement branches of the gene family. Additionally CD109 carries the antigenic determinant of the Gov alloantigen system, which has been implicated in a subset of immune mediated platelet destruction syndromes. In this thesis, the status of CD109 in the evolution and phylogeny of the A2M family has been clarified. First, I elucidated the evolutionary relationships of CD109, and of the other eight human A2M/C3/C4/C5 proteins, using sequence analysis and a detailed comparison of the organization of the corresponding loci. Extension of this analysis to compare CD109 to related sequences extending back to placazoans, defined CD109 as a member of a distinct and archaic branch of the A2M phylogenetic tree. Second, in conjunction with collaborators, the molecular basis of the Gov alloantigen system was identified as an allele specific A2108C; Y703S polymorphism. Utilizing cDNA and genomic sequence we then developed methods to accurately and precisely genotype the Gov system. Finally, the expression kinetics of platelet CD109 was elucidated, in order to obtain basic information regarding its expression and subcellular localization, and to resolve discrepancies in reported platelet CD109 expression. Quantitative flow cytometry demonstrated that CD109 was expressed on the surface of activated platelets at very low levels in most healthy volunteers. In resting platelets, CD109 was localized to the OCS and intracellular storage granules. CD109 displayed differential agonist induced expression in comparison to GPIIb/IIIa epitope unmasking, and surface expression of CD62P and CD63. CD109 was rapidly expressed on the cell surface in response to low doses of both strong and weak agonists. This early expression is likely the result of CD109’s proximity to the plasma membrane in resting platelets. As such, CD109 is positioned to participate at early stages of primary haemostasis.

CD109 is a 170kD glycosylphosphatidylinositol-anchored protein expressed on subsets of fetal and adult CD34+ haematopoietic stem cells, endothelial cells, activated T cells, and activated platelets. Cloning of the CD109 cDNA by our group identified the molecule as a novel member of the alpha2M/C3/C4/C5 family of thioester containing proteins. Curiously, CD109 bears features of both the alpha2M and complement branches of the gene family. Additionally CD109 carries the antigenic determinant of the Gov alloantigen system, which has been implicated in a subset of immune mediated platelet destruction syndromes. In this thesis, the status of CD109 in the evolution and phylogeny of the A2M family has been clarified. First, I elucidated the evolutionary relationships of CD109, and of the other eight human A2M/C3/C4/C5 proteins, using sequence analysis and a detailed comparison of the organization of the corresponding loci. Extension of this analysis to compare CD109 to related sequences extending back to placazoans, defined CD109 as a member of a distinct and archaic branch of the A2M phylogenetic tree. Second, in conjunction with collaborators, the molecular basis of the Gov alloantigen system was identified as an allele specific A2108C; Y703S polymorphism. Utilizing cDNA and genomic sequence we then developed methods to accurately and precisely genotype the Gov system. Finally, the expression kinetics of platelet CD109 was elucidated, in order to obtain basic information regarding its expression and subcellular localization, and to resolve discrepancies in reported platelet CD109 expression. Quantitative flow cytometry demonstrated that CD109 was expressed on the surface of activated platelets at very low levels in most healthy volunteers. In resting platelets, CD109 was localized to the OCS and intracellular storage granules. CD109 displayed differential agonist induced expression in comparison to GPIIb/IIIa epitope unmasking, and surface expression of CD62P and CD63. CD109 was rapidly expressed on the cell surface in response to low doses of both strong and weak agonists. This early expression is likely the result of CD109’s proximity to the plasma membrane in resting platelets. As such, CD109 is positioned to participate at early stages of primary haemostasis.

Surfactant protein D (SP-D) is an important innate immune collectin involved in uptake and clearance of microbes and allergens in the lungs. SP-D has been shown to ameliorate allergic asthma reactions in mice; however, the mechanisms for this are not fully understood. We investigated the role of SP-D in the uptake and clearance of the model allergen ovalbumin (OVA) by macrophages. We discovered that SP-D does not bind OVA but binds fractions with contaminating proteins; ovomucin and ovomacroglobulin. We extended these findings to show that SP-D binds human alpha-2-macroglobulin (A2M) in its cleaved or intact state, in a concentration-, calcium-, and carbohydrate-dependent manner. A2M increases the innate immune potential of SP-D by increasing its ability to agglutinate the bacteria Escherichia coli and Bacillus subtilis. We found that SP-D does not increase the uptake of OVA by murine macrophage cell lines, or by alveolar macrophages in vivo in BALB/cJ mice.

Surfactant protein D (SP-D) is an important innate immune collectin involved in uptake and clearance of microbes and allergens in the lungs. SP-D has been shown to ameliorate allergic asthma reactions in mice; however, the mechanisms for this are not fully understood. We investigated the role of SP-D in the uptake and clearance of the model allergen ovalbumin (OVA) by macrophages. We discovered that SP-D does not bind OVA but binds fractions with contaminating proteins; ovomucin and ovomacroglobulin. We extended these findings to show that SP-D binds human alpha-2-macroglobulin (A2M) in its cleaved or intact state, in a concentration-, calcium-, and carbohydrate-dependent manner. A2M increases the innate immune potential of SP-D by increasing its ability to agglutinate the bacteria Escherichia coli and Bacillus subtilis. We found that SP-D does not increase the uptake of OVA by murine macrophage cell lines, or by alveolar macrophages in vivo in BALB/cJ mice.

The thesis "The proteomic analysis in hematology: Identification of alfa2- macroglobulin as a specific carrier for the hormone hepcidin and proteomic analysis of the leukemic K562 cell differentiation induced by sodium butyrate" describes proteomic approaches, used for the identification and functional characterisation of proteins, which are binding and transporting the iron metabolism regulating hormone hepcidin. Proteomic techniques are also exploited for the identification of proteins, participating in erythroid differentiation of the model cell line K562. In the first section of the thesis, non-denaturing, native techniques, such as chromatography and native electrophoresis are used, in the second section, the control and butyrate - induced K562 cell proteomes are compared using the classical 2D - SDS polyacrylamide gel electrophoresis approach. The methods, described in the thesis are broadening the spectrum of available techniques in experimental hematology. The results, described in this thesis together with the accompanying published manuscripts broaden our knowledge in the function of proteins of iron metabolism and proteins, functioning in erythroid differentiation. Key words: proteomic analysis, hepcidin, alfa2-macroglobulin, iron metabolism, CML, K562, sodium butyrate

Diabetic kidney disease (DKD) is the leading cause of end stage renal disease worldwide and occurs in up to 40% of patients with diabetes. The standard of care for DKD treatment has not kept up with the current health epidemic, which has led to a heavy economic toll and substantial health burden. Targeting either cell surface (cs)GRP78, activated α2-macroglobulin (α2M*) or preventing their interaction may provide a novel anti-fibrotic therapeutic target for the treatment of DKD and potentially non-diabetic chronic kidney disease (CKD) as well. Previously our lab has shown that HG-induced csGRP78 is a mediator of PI3k/Akt signaling and downstream extracellular matrix (ECM) protein production in glomerular mesangial cells (MC). However, the ligand responsible for activating high glucose (HG)-induced csGRP78 had not yet been determined. We have shown thus far that α2M is endogenously produced, secreted, and activated (denoted α2M*) in HG by MC, which leads to its binding to and activation thereof csGRP78. Further, α2M knockdown or α2M* neutralization attenuated Akt activation, the production of the profibrotic cytokine connective growth tissue factor (CTGF) and ECM proteins fibronectin and collagen IV. We have also shown that integrin β1 (Intβ1), a transmembrane receptor, associated with csGRP78 under HG conditions and likely acts as a tether to present csGRP78 completely extracellularly on MC. Interestingly, Intβ1 activation, even in the absence of HG, was sufficient to induce csGRP78 translocation. Further, inhibition of either csGRP78 or Intβ1 prevented synthesis, secretion and signaling of TGFβ1. This data implicates a role for Intβ1 as a required signaling partner for csGRP78-mediated profibrotic signaling. To further our understanding of csGRP78/ α2M*’s role in DKD, we investigated their ability to mediate TGFβ1 signaling through its non-proteolytic activator thrombospondin-1 (TSP1). Here, HG-induced TSP1 expression, ECM deposition, and activation of TGFβ1 was regulated by the PI3k/Akt pathway via csGRP78/α2M* in MC. Furthermore, we assessed whether this csGRP78/ α2M* axis is relevant to promoting profibrotic signaling in other renal cell types, including proximal tubule epithelial cells (PTEC) and renal fibroblasts (RF), that contribute to the pathogenesis of both later stage DKD and non-diabetic CKD. We show evidence here that HG and direct treatment with TGFβ1, a key pathologic regulator of kidney fibrosis, induce GRP78 surface translocation as well as the endogenous production and activation of α2M in both PTEC and RF. Inhibition of either csGRP78 or α2M* prevented TGFβ1 signaling measured as Smad3 activation as well as downstream ECM production. Interestingly, inhibition of this pathway under direct TGFβ1 treatment did not prevent Smad3 activation, implicating a role for Smad-independent TGFβ1 signaling through this axis. We identified the known noncanonical TGFβ1 signaling partners, yes associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ), are mediated by csGRP78 and α2M*. Lastly, we evaluated the potential therapeutic benefit of inhibiting csGRP78/α2M* interaction in the kidney fibrosis model, unilateral ureteral obstruction (UUO). Here, we show evidence that inhibition of this signaling axis using an inhibitory peptide can prevent renal fibrosis. Whether this peptide also prevents fibrosis in DKD is currently being assessed. Together, these studies strongly implicate targeting csGRP78/α2M* interaction as a novel anti-fibrotic therapeutic intervention for early and late stage DKD, as well as a potential role in non-diabetic CKD. / Thesis / Doctor of Philosophy (Medical Science) / Diabetic kidney disease is the leading cause of kidney failure in developed nations. This progressive disease leads to the loss of kidney function due to an accumulation of scar proteins in the kidney over time. High glucose is a major factor that causes this to occur. Our lab studies specific kidney cells called mesangial cells, proximal tubule epithelial cells, and fibroblasts that produce scar proteins in the presence of high glucose. We have shown that when these cells are treated with high glucose, this causes the movement of a protein called GRP78 that normally resides inside the cell to move to the cell’s surface where it can interact with other proteins. My research has established that the proteins alpha 2-macroglobulin (ɑ2M), integrin β1 (Intβ1), and thrombospondin-1 (TSP1) can bind to GRP78 on the cell surface and cause cells to make scar proteins. Preventing ɑ2M or Intβ1 from binding to GRP78 or preventing TSP1 production prevents mesangial cells from making scar proteins when exposed to high glucose. In a mouse model that overproduces these scar proteins, we showed that preventing cell surface GRP78 and α2M interaction prevents scar protein production and is thus a novel potential treatment option for kidney disease.