ABSTRACT STEVEN M. JOHNSON Use of Click Chemistry to Assess Glycoprotein Dynamics in Cultured Cells. (Under the Direction of RICHARD STEET, PH.D.) Bioorthogonal chemical reporters are novel reagents in the field of chemical glycobiology that can be used for in vivo imaging of glycoproteins in cultured cells. Unlike large, bulky genetically encoded tags such as green fluorescent protein (GFP), these reporters are small molecules that can be incorporated into the cell s glycan biosynthetic machinery without harming the biological system. One such reporter, N-azidoacetylmannosamine (ManNAz), is a sugar analog that is capable of integrating into nascent glycoproteins as sialic acid residues. A chemical handle can then be covalently linked to the azido group of incorporated ManNAz residues, allowing for the visualization of the modified glycoproteins using fluorescent probes that recognize this handle. Our goal is to use this technique (known as click chemistry ) to investigate the nature and dynamics of glycoprotein storage in lysosomal storage disorders such as mucolipidosis II (ML-II), a congenital disease characterized by improper catabolism and accumulation of undigested macromolecules. Employing both epifluorescence and confocal microscopy, we have successfully visualized the accumulation of sialic acid-containing glycoproteins in the lysosomes of ML-II cells. Furthermore, the click chemistry has allowed us to view the dynamics of extracellular matrix (ECM) glycoprotein synthesis and turnover within WT cells. The application of different azide sugar precursors in other lysosomal storage disease tissues should provide us with additional ways to study the trafficking and turnover of glycoproteins in these cells. Our findings could facilitate new methods for lysosomal disease diagnosis and will provide unprecedented opportunities to track glycoprotein dynamics and turnover in affected cell types. INDEX WORDS: Epifluorescence, Glycoproteins, Lysosomal Storage Disorders, Lysosome, Mucolipidosis-II, Congenital Disorders of Glycosylation
USE OF CLICK CHEMISTRY TO ASSESS GLYCOPROTEIN DYNAMICS IN CULTURED CELLS by STEVEN M. JOHNSON A Thesis Submitted to the Honors Council of the University of Georgia in Partial Fulfillment of the Requirements for the Degree BACHELOR OF SCIENCE in BIOCHEMISTRY AND MOLECULAR BIOLOGY with HIGH HONORS and CURO SCHOLAR DISTINCTION Athens, Georgia 2009
2009 Steven M. Johnson All Rights Reserved
USE OF CLICK CHEMISTRY TO ASSESS GLYCOPROTEIN DYNAMICS IN CULTURED CELLS by STEVEN M. JOHNSON Approved: Richard Steet, Ph.D. July 27, 2009 Dr. Richard Steet Date Faculty Research Mentor Approved: Harry Dailey, Ph.D. July 27, 2009 Dr. Harry Dailey Date Reader Approved: David Williams, Ph.D. July 31, 2009 Dr. David Williams Date Director, Honors Program, Foundation Fellows and Center for Undergraduate Research Opportunities Approved: Pamela Kleiber, Ph.D. July 31, 2009 Dr. Pamela B. Kleiber Date Associate Director, Honors Program and Center for Undergraduate Research Opportunities
ACKNOWLEDGEMENTS I would like to thank the entire Steet Laboratory at the Complex Carbohydrate Research Center, especially my faculty research mentor Dr. Richard Steet who helped guide me in the right direction for the duration of this project. I am also very grateful to Dr. Heather Flanagan- Steet for assistance with confocal microscopy, members of the Steet laboratory for helpful discussions and my reader Dr. Harry Dailey for his insightful suggestions. I also wish to thank members of Geert-Jan Boons laboratory for generating key reagents and Dr. Mark Haskins for providing feline ML-II cell samples. This work was supported by start-up funds (R.S.) and a grant from the Mallinckrodt Foundation (R.S.). iv
TABLE OF CONTENTS ACKNOWLEDGEMENTS... iv LIST OF FIGURES... vi CHAPTERS Page 1 INTRODUCTION AND BACKGROUND...1 Cellular Biology of the Lysosome...1 Lysosomal Storage Disorders...2 Click Chemistry Methodology...4 2 MATERIALS AND METHODS...9 3 CHEMICALLY-INDUCED GLYCOPROTEIN STORAGE IN WILD TYPE CELLS...11 4 GENETICALLY-INDUCED GLYCOPROTEIN STORAGE IN DISEASED CELLS...13 Genetically-Induced Storage in ML-II Human Skin Fibroblasts...13 ML-II Feline Connective Tissue Samples Show Storage and Improper Protein Turnover...15 5 ANALYSIS OF PROTEIN TURNOVER IN WT AND DISEASED CELLS...18 Impaired Turnover in Human ML-II Cells...20 Neuraminidase-Deficient Human Fibroblasts...22 6 CONCLUSIONS...24 7 WORKS CITED...27 v
LIST OF FIGURES Page Figure 1: Proper Sorting of Lysosomal Enzyme in WT Cells...3 Figure 2: Missorting of Lysosomal Enzyme in ML-II Due to Defect in Phosphotransferase...3 Figure 3: Incorporation of Sugar into Glycoconjugates Inside a Golgi Compartment...5 Figure 4: ManNAz Incorporation as Sialic Acid Residues...6 Figure 5: Step 1 of the Click Chemistry...7 Figure 6: Step 2 of the Click Chemistry...8 Figure 7: Chloroquine Treatment...12 Figure 8: Genetically-Induced Storage in ML-II Human Skin Fibroblasts...14 Figure 9: Feline Synovial Fibroblasts Exhibit Storage of ECM Protein...16 Figure 10: Lysosomal Storage in ML-II Feline Chondrocyte...17 Figure 11: ECM Turnover Can Be Monitored Following Washout of ManNAz in WT Cells...19 Figure 12: Impaired Turnover of Glycoproteins Visualized in ML-II Human Fibroblasts...21 Figure 13: Altered Trafficking and Turnover in Neuraminidase-Deficient Fibroblasts...23 vi
CHAPTER 1 INTRODUCTION AND BACKGROUND Cellular Biology of the Lysosome The cell is the dynamic, ever-adapting building block of all living material. It consists of a multitude of organelles, each with unique duties to carry out the processes necessary for sustainable life. The lysosome, the organelle responsible for the recycling and catabolic digestion of macromolecules, is vital in maintenance of cellular homeostasis, and its functions are multi-faceted. The lysosome is the component of the cell that breaks down ingested material (i.e. invading microbes, other dying cells) from phagocytosis. It also performs the necessary process of recycling within the cell. Receptor proteins, old organelles, or most forms of unneeded or excess cell waste can be catabolized by the enzymes of the lysosome into their base components. These resultant building blocks can then be recycled into nascent products needed by the cell. 1 The ph of the interior of the lysosome is a striking feature of this organelle. It is around 4.8 whereas the ph of the surrounding cytosol is roughly 7.3 (slightly basic). 1 Because the digestive enzymes (hydrolases, proteases, etc.) contained by the lysosome require an acidic environment for optimum function, this ph difference is important because it prevents any rogue digestion of necessary macromolecules in other parts of the cell should a lysosomal enzyme leak from the lysosomal membrane. This organelle truly is a dynamic and ever-important component of the cell. It can be seen that any malfunction of the lysosome would be disastrous for the cell and the organism as a whole. 1
Lysosomal Storage Disorders Lysosomal storage disorders (LSDs) are a group of inherited genetic diseases caused by defects in the breakdown of macromolecules within lysosomes, the disposal and recycling compartment of the cell. Since lysosomes contain digestive enzymes (i.e. acid hydrolases) that function to degrade macromolecules (such as glycoproteins), defective activity in these enzymes leads to storage of undegraded macromolecules within this organelle. Though much is known about the genetic basis of LSDs, the pathophysiology of these disorders (the mechanisms whereby storage causes disease symptoms) remains poorly understood. 2 Several studies have indicated that intracellular trafficking events are disrupted in cells from LSD patients 3 but details of these effects are not currently understood, and it is clear that techniques to address this issue are needed. Thus, the main question needing to be addressed is: How is glycoprotein trafficking and turnover affected in lysosomal storage disorders? To attempt to answer this question, we are exploring the use of click chemistry, a novel chemical fluorescent tagging technique discussed below, to monitor glycoprotein turnover and storage in LSD cells using mucolipidosis II (ML-II or I-cell disease) as our primary disease model. The disease is characterized by a defect in the phosphotransferase enzyme that catalyzes the first step in the biosynthesis of mannose-6-phosphate (M6P) residues on glycans of most soluble lysosomal hydrolases. 3 M6P is the sugar-based recognition marker needed for the proper targeting of most soluble hydrolases to the lysosome (see Figure 1 below). Without this M6P targeting marker, the enzymes are improperly sorted (see Figure 2 below), leading to their subsequent secretion to the extracellular space. 2
Mannose-6 Phosphorylation ER Golgi Lysosome Figure 1: Proper Sorting of Lysosomal Enzyme in WT Cells. The lysosomal protein (indicated by the red triangle) is synthesized by a ribosome in the endoplasmic reticulum (ER) and sent to the Golgi where the mannose-6-phosphate lysosomal targeting marker is added, resulting in trafficking of the enzyme to the lysosome. Defective Phosphotransferase ER Golgi Lysosome Figure 2: Missorting of Lysosomal Enzyme in ML-II Due to Defect in Phosphotransferase. The lysosomal protein (indicated by the red triangle) is synthesized by a ribosome in the endoplasmic reticulum and sent to the Golgi where defective glycosylation occurs, resulting in secretion of the enzyme by the cell. 3
There is evidence that ML-II cells, particularly those involved in connective tissue homeostasis, possess numerous cytoplasmic inclusions and/or dense lysosomes containing uncatabolized macromolecules. The characterized inclusions are presumed to be caused by the lack of hydrolytic enzymes (e.g. glycosidases and cathepsin proteases) within the lysosome. The following describes click methodology that was developed (described in Methodology ) and employed to gauge the extent and dynamics of glycoprotein turnover and storage in various WT and LSD cells. Hopefully, the use of click chemistry in these cell lines will provide a key first step in assessing the pathomechanisms of ML-II and other LSDs. Click Chemistry Methodology Since the invention of new reagents that allow for easier visualization of the cell, the area of chemical glycobiology has shown great importance in furthering the study of diseases. The study of glycoproteins is important for understanding the roles certain organelles play in the complex biological processes of which cells are apart. Therefore, being able to visualize these glycoproteins is extremely important. Usually, genetically encoded fluorescent labels (such as GFP) are used for visualizing proteins. But these large tags can cause structural changes to the protein and are not ideal for glycan study. 4 Methods are continually being developed that allow for the visualization of cellular components in vivo and one of these, click chemistry, is relatively easy and safe to the studied cell line. Through the synthesis of an array of sugars that can be incorporated into nascent glycoproteins, it is possible to analyze the dynamics of glycan activity in an unparalleled fashion. The click reagents are groundbreaking in that they circumvent the problem of size; the new approach involves the introduction of a small functional group to the glycoprotein using the cell s own biosynthetic machinery. The functional group (an azido group 4
in these experiments) is useful because it is inert in a biological environment and is extremely rare in biological systems. 4 This group (also called a bio-orthogonal chemical reporter) is chemically reactive and can be used to covalently attach fluorescent tags and, thereby, allowing for the visualization of the object of study inside cells and/or the extracellular matrix. 5 The process of attaching probes to these reporters involves the cell s normal biosynthetic machinery (summarized in Figure I). Azido sugars fed to cells are first converted into highenergy nucleotide-sugars within the cytosol. These donors are then brought into the Golgi apparatus by specific nucleotide-sugar transporters where they are utilized by glycosyltransferases that integrate the azido sugars into glycoconjugates (see Figure 3 below). nucleotide- - sugar cytosol transporter lumen nucleotide- nucleotide glycosyltransferase Figure 3: Incorporation of Sugar into Glycoconjugates Inside a Golgi Compartment 5
The overall function of the machinery goes unaltered by the azido sugar (Nazidoacetylmannosamine (ManNAz) in these experiments). In order to enhance delivery and uptake of ManNAz into the cells, these monosaccharides are typically acetylated to allow facile transport across the hydrophobic plasma membrane. Once inside the cell, however, non-specific esterases remove the acetyl groups to generate the precursor sugars (see Figure 4 below). figure adapted from Invitrogen website AcO AcO AcO outside of cell O HN O N 3 OAc AcO AcO AcO O HN O N 3 OAc nonspecific esterases Ac 4 ManNAz Ac 4 ManNAz O N 3 N 3 HO O OH OH H N H O CO 2 - O SiaNAz on cellsurface glycan incorporated into the sialic acid biosynthetic pathway HO HO HO HN O ManNAz OH Figure 4: ManNAz Incorporation as Sialic Acid Residues The attached azido sugars can then be labeled with an exogenously supplied fluorescent probe, leading to the ability to visualize the cells using basic fluorescent microscopy. In the current work, this was accomplished by exposing the cells with the azide-linked glycoproteins to a biotin molecule containing a terminally attached alkyne (Cmpd #188) for 1 hour. The alkyne and azide chemically react to form a (3+2) cycloaddition product which attaches the glycoprotein to the biotin molecule 6 (see Figure 5 below). 6
Glycoprotein N N N N 1 hour Room temperature N N Biotin Compound #188 (biotin alkyne) Stable Triazole Conjugate Figure 5: Step 1 of the Click Chemistry Azido-labeled glycoprotein undergoes reaction with biotin alkyne, forming a stable triazole conjugate to be reacted in the next step. Cells now possessing the biotin-linked glycoproteins are then exposed to an avidinfluorophore secondary antibody (streptavidin-568), exploiting the strong affinity between avidin and biotin to attach the fluorescent probe to the glycoprotein (see Figure 6 below). The glycoproteins can then be visualized under a fluorescent microscope and their subcellular localization investigated. 7
Streptavidin Streptavidin-568 N N N Stable Triazole Conjugate 30 min. Room temperature Fluorophore-conjugated glycoprotein Figure 6: Step 2 of the Click Chemistry Stable Triazole Conjugate undergoes reaction with Streptavidin-fluorophore to form the fluorophore-conjugated glycoprotein. 8
CHAPTER 2 MATERIALS AND METHODS The protocol used for ALL staining experiments follows. Cells were grown for 2-3 days as needed in DMEM media with 10% Fetal Bovine Serum in 100 u/ml pen-strep at 37 C and 5% CO 2. Cells were then plated on coverslips and cultured overnight in a 12-well plate. The cultures were incubated at 37 C and 5% CO 2 with the azido sugar analog ManNAz for 1 day at a final concentration ranging from 2.5µM to 50µM. The azido sugar analog is utilized by the sialic acid biosynthetic machinery, thereby resulting in a cell-surface glycan with an attached azido group. The cells were incubated with a 30µM solution of Compound #188 (in Dulbecco s phosphate buffered saline (DPBS) with Ca ++ and Mg ++ ), which consists of the biotin molecule with a terminal alkyne group, for 1 hour at room temperature (RT). The terminal alkyne reacts with the azido group in a cycloaddition reaction. After the tagging process, the cells were fixed in 10% formaldehyde in phosphate buffered saline (PBS) for 15 minutes at RT. Then, a 2µg/mL solution of Alexa Fluor Streptavidin-568 (fluoresces red) in PBS with 10% bovine serine albumin (BSA) and 0.02% Triton X-100 (T-100) was added to complete the click chemistry reactions. The cells were then washed 3 times for 10 minutes at RT in PBS to remove excess and unconjugated streptavidin-568. Coverslips were then mounted onto glass slides with Prolong Gold mounting media. In addition to the azido sugar labeling, the majority of the experiments involved costaining with an additional antibody tag to track the azido-labeled glycoprotein. Throughout the course of the project, many different intracellular compartments (lysosomes, endosomes, etc.) were labeled to determine possible co-localization with the azido-labeled glycoprotein. This 9
involves the following process. After the cells were fixed and incubated with Compound #188 as described above, a primary antibody (in PBS with 10% BSA and 0.02% T-100) targeting the specified protein is added to the wells for 1 hour at RT. The cells are then washed 3 times for 10 minutes in PBS at RT. Following washes, a secondary antibody (in PBS with 10% BSA and 0.02% T-100) with a conjugated fluorophore is added. When labeling with streptavidin-568, which fluoresces red, a 488-conjugated secondary antibody (which fluoresces green) was employed as a co-stain to determine possible colocalization. 10
CHAPTER 3 CHEMICALLY-INDUCED GLYCOPROTEIN STORAGE IN WILD TYPE CELLS Chloroquine (N'-(7-chloroquinolin-4-yl)-N,N-diethyl-pentane-1,4-diamine) is a drug that has long been used in the treatment of malaria. 7 The drug slightly raises intralysosomal ph, causing accumulation of the chemical within the lysosomal membrane. Because lysosomal hydrolases and other enzymes are trafficked to the lysosome from the Golgi based on a ph gradient, nascent glycoproteins destined for lysosomal function are missorted, and, thus, not imported into the lysosome. Therefore, lysosomal catabolism cannot be properly achieved. Thus, upon exposure of healthy wild-type cells to chloroquine and ManNAz, it is possible to view chemically-induced lysosomal storage under the fluorescent microscope. Healthy human skin fibroblasts were treated with 25µM ManNAz (and 25µM ManNAc as a control) for 1 day and then given a 2-day washout period (at 37 C) in which new media was added without the azido sugar. Select cultures were then treated with 50µM chloroquine for 1 hour. The chloroquine-treated cells were also exposed to a 1:1000 rabbit cathepsin D (a known lysosomal aspartyl protease that is unaffected by the chloroquine treatment) antibody dilution to monitor lysosomal storage (see Figure 7 below). Wild-type cells that were not treated with chloroquine (A) show extracellular matrix staining (this will be discussed in later chapters) and intracellular glycoprotein labeling by the ManNAz reagent. The control (C) proved to be effective, as there was little staining compared with the ManNAz. It can be seen from the overlap of the chloroquine-labeled cells (B) and the cathepsin D costain (D) that the storage induced by the chloroquine was in fact lysosomal. These results proved useful in that chemically-induced lysosomal storage (as opposed to the 11
genetically-induced lysosomal storage caused by congenital lysosomal storage disorders) could be produced via exposure to the drug. This information provided the basis for determining the extent of lysosomal storage in the future experiments involving Mucolipidosis-II (ML-II) diseased cells. The storage exhibited by the ML-II cells stained extremely similarly to those WT cells treated with chloroquine. A B WT ManNAz WT ManNAz + Chloroquine C D WT ManNAc WT Cathepsin D Figure 7 - Chloroquine Treatment: Note that accumulation of ManNAz-labeled glycoproteins in intracellular vesicles following treatment with Chloroquine; this accumulation is reminiscient of the dense lysosomes found in lysosomal storage diseased cells. 12
CHAPTER 4 GENETICALLY-INDUCED GLYCOPROTEIN STORAGE IN DISEASED CELLS Because the treatment of cells with the drug chloroquine allowed for visualization of chemically-induced storage, reproduction of similar storage genetically was needed to anchor the usefulness of the azido click chemistry. Therefore, using the Mucolipidosis-II model, diseased cells were treated with ManNAz and other costains and qualitatively examined. In addition to WT and ML-II human skin fibroblasts lines, samples of feline ML-II tissues which were generously provided by Dr. Mark Haskins of the University of Pennsylvania were available for study. Genetically-Induced Storage in ML-II Human Skin Fibroblasts ML-II human skin fibroblasts were continuously fed 25 µm ManNAz for 1 day, followed by the labeling process with the click chemistry. The results of this experiment revealed massive lysosomal accumulation of material in ML-II and only a small amount in the WT cells, most likely due to the ongoing process of proper degradation (see Figure 8 below). There was also more stained extracellular matrix (ECM) in the WT cells than the ML-II cells. This is significant insofar as it could be shown that the glycoproteins being labeled by the ManNAz were probably extracellular matrix proteins. It is unclear which ECM proteins are labeled with the click chemistry, however, in later experiments it is shown that there was a strong correlation between the ManNAz staining pattern and that of fibronectin, a known ECM protein (see next section). Because the lysosomal enzymes in the WT cells are functioning properly, protein turnover (degradation/recycling) is correctly managed by the cell (more about defective protein 13
turnover will be discussed in subsequent sections). In the ML-II cells, however, ECM proteins are most likely being endocytosed and sent to the lysosome as a part of turnover, but no degradation is occurring because of the improperly functioning and missorted digestive enzymes. This experiment was also significant in that it provided similar results (click-labeled lysosomal storage) to the chloroquine-treated WT cells, indicating that there is most likely a problem with the lysosomes in these cells. WT ML-II Figure 8: Genetically-Induced Storage in ML-II Human Skin Fibroblasts Note the dense lysosomal inclusions in the ML-II fibroblasts consistent with the accumulation of undigested macromolecules within the lysosome. This accumulation is strikingly similar to the phenotype of the chloroquine-treated cells. Also note the ECM staining in the WT cells. The stringy, continuous staining pattern (see arrow) is indicative of extracellular matrix. 14
ML-II Feline Connective Tissue Samples Show Storage and Improper Protein Turnover Though there are a multitude of clinical effects plaguing ML-II patients, a substantial amount of damage occurs in the craniofacial region and in connective tissues. Therefore, the experiments involving the feline ML-II chondrocytes and synovial fibroblasts (crucial players in connective tissue homeostasis) proved to be insightful. Since few antibodies are available to study a feline system (most are monoclonal or polyclonal from mice or rabbit) using the click chemistry circumvents this problem and allows for examination of the dynamics of ML-II in the cat samples with a non-biased tracking tool. The results of the staining of the synovial fibroblasts provided evidence of the value of the reagent and advanced the lab s goal of understanding ML-II disease-specific dynamics. Because ML-II patients have significant damage to cartilaginous areas of the body, examining storage within diseased synovial fibroblasts should prove to be insightful. It can be seen from Figure 9 that the WT cells have very high amounts of intact extracellular matrix (as indicated by the Fibronectin stain) and little lysosomal storage. ML-II synovial fibroblasts, however, show progressive lysosomal storage (see arrow) and less intact ECM. It is also important to note the yellow oval in the ML-II cells. In the designated region there are hints of a shade of yellow, signifying colocalization of the two secondary antibodies. This is indicative that fragments of undigested fibronectin and ManNAz-labeled glycoprotein are present in the same vesicle (or at least in a very similar location within the cell). Therefore, it may be assumed that ML-II cells are deficient in the turnover of ECM proteins. This was significant in that it is possible to see first-hand improper extracellular matrix turnover within cells specific to connective tissue and cartilage homeostasis. 15
WT ML-II Fibronectin ManNAz Figure 9: Feline Synovial Fibroblasts Exhibit Storage of ECM Protein WT and ML-II feline synovial fibroblasts were labeled with 25 μm ManNAz and co-stained for fibronectin (a rabbit antibody in a 1:1000 dilution), a known extracellular matrix protein. Note the colocalization of ManNAz and fibronectin (see yellow oval) in ML-II cells; this is not present in the WT cells, indicating that ML-II cells are improperly degrading matrix proteins such as fibronectin. Data from the staining of feline chondrocytes provided additional support for what was found in the synovial fibroblasts. Though chondrocytes are different from synovial fibroblasts (chondrocytes exist in cartilage and synovial fibroblasts are contained in the synovial fluid of joints), the results of the experiment were still positive in that lysosomal storage could be shown. Though no other tissues from the feline samples were tested with the click chemistry, the 16
labeling tool has been valuable for documenting the storage within these cell types so important in connective tissue. See Figure 10 and notice the lysosomal storage within the chondrocytes. Figure 10: Lysosomal Storage in ML-II Feline Chondrocyte Note the lysosomal storage (see arrow). Because we now have evidence of lysosomal storage in two types of the feline connective tissue via the click chemistry, this novel technique has proven sufficient in documenting new data regarding ML-II. 17
CHAPTER 5 ANALYSIS OF PROTEIN TURNOVER IN WT AND DISEASED CELLS Because protein turnover within the cell via the lysosome is a dynamic process that is constantly occurring, it is necessary to study the progression of click chemistry-labeling on different time scales rather than restricting the analysis to only one time point. Thus, pulse-chase experiments and experiments involving washouts were implemented to compare the rate of turnover of click-labeled protein within the cell. This allowed for visualization of the cells at different time points, giving insight into the turnover process as a whole. For the washout experiment, one set of WT cells was continuously labeled with ManNAz for 24 hours, leaving the cells constantly exposed to the azido sugar. Another set of cells were fed the ManNAz for 6 hours and then given new media without the azido sugar for the remaining 18 hours (see Figure 11 below). ECM protein turnover can be tracked with the click chemistry in this fashion. The stringy, continuous strands have been somewhat diminished in the washout cells, indicating that the matrix containing the click-labeled glycoproteins is getting replaced with new ECM proteins. Therefore, this shows that the click chemistry is effective in tracking protein turnover within the cell. 18
24 hr labeling 6 hr labeling, 18 hr washout Figure 11: ECM Turnover Can Be Monitored Following Washout of ManNAz in WT Cells WT Human Fibroblasts have been continuously labeled with 25 μm ManNAz for 24 hours (left) and for 6 hrs and then given new media (a washout period) for 18 hrs (right). Note the breaks in the staining of the extracellular matrix (see arrows). This is indicative of the turnover of labeled extracellular matrix proteins and replacement with new, unlabeled protein. There were two experiments performed using the pulse-chase method, one slightly different than the ManNAz washouts described above. In these experiments, the cells were fed 50µM ManNAz for 1 day, pulsed (exposed to) with the biotin-alkyne Compound #188 for 1 hour, and then chased (given new media without #188) for different lengths of time (see Figure 19
12 below). Examining the process in this manner is the first step in uncovering the pathogenesis and mechanisms of the trafficking and storage problems that are apparent in these cells. Impaired Turnover in Human ML-II Cells In the first pulse-chase experiment human WT and ML-II skin fibroblasts were examined. It can be seen from Figure X that the protein turnover process is impaired. Comparing the t 0 of the WT and ML-II cells (A and D, respectively), one can see a small difference between the two. There appears to be less accumulated lysosomal storage in the WT versus the ML-II. In the 4 hr. chase of the WT and ML-II samples (B and E, respectively), however, the difference becomes substantial. The data for the WT cell shows normal catabolic processing within the lysosome, breaking down the ManNAz-linked glycoproteins. However, the ML-II cell seems to have excess storage that results from an inability to break down the endocytosed glycoproteins. It can be clearly seen that there is more of the Compound #188 left in the ML-II cell versus the WT even when the two had a similar amount of staining in the t 0. Another interesting feature about this experiment were the results obtained in experiments that employed the costain with LysoTracker, a dye that tracks acidic vesicles within the cell. Because lysosomes are the major acidic vesicles (some late endosomes become acidic when nearing fusion with lysosomes), lysosomes can be easily tracked. In the WT cell (block C), these is normal lysosomal activity. In the ML-II cell, however, there seems to be a mass proliferation of lysosomes. Though the cause of this proliferation is unclear, it is most likely due to the cell trying to counter the problem of undigested macromolecules and secreted missorted lysosomal enzymes. 20
A No Chase B 4 Hr Chase C LysoTracker WT D E F ML-II Figure 12: Impaired Turnover of Glycoproteins Visualized in ML-II Human Fibroblasts WT and ML-II human fibroblasts were labeled with 50 μm ManNAz and pulsed with Cmpd. #188 and then chased for 0 hrs (t 0 left column) and 4 hrs. (middle column). The right column shows cells labeled for 15 minutes with a 1:100,000 dilution of LysoTracker in PBS. After exposure to the dye, cells were immediately fixed and mounted. Note the difference in turnover between t 0 and the 4 hr chase. More ManNAz is retained as storage in the ML-II cells. Also, the large increase in staining of LysoTracker in ML-II cells indicates overproduction of lysosomes in attempt to cope with the problem of undigested material. 21
Neuraminidase-Deficient Human Fibroblasts Because the click chemistry proved to be effective for exploring the dynamics of Mucolipidosis-II, we thought it could be useful in examining other diseases. A relatively unstudied congenital disorder of glycosylation (CDG) involves neuraminidase deficiency (ND). Neuraminidase is a hydrolase that cleaves the glycosidic linkages of sialic acid. Therefore, the ManNAz click chemistry was perfect for viewing the effects of trafficking and turnover of sialic acid in these cells where it is impossible to completely breakdown sialic acid (at least, by the neuraminidase pathway). Comparing the t 0 ( no chase ) of the WT and ND cells (see Figure 13 below), there is little difference in the staining pattern. It is mostly cell surface, similar to the pattern in the last experiment. In the 1 hr. chase, both the WT and ND cells show successful trafficking to the lysosome. In the 4 hr. chase, however, is where the main difference lies. The WT, as shown before, has properly trafficked the sialic acid residues to the lysosome for degradation and the digestive enzymes have effectively catabolized the macromolecules, seeing how only very faint staining remains. In the ND cells, the sialic acid residues have been trafficked to the lysosome, but the lack of the properly-functioning neuraminidase has led to a build up of the sialic acid residues within the compartment. The data from this experiment show an example of proper sialic acid trafficking and turnover in addition to defective sialic acid trafficking and turnover. 22
No chase 1 hr. chase 4 hr. chase WT ND Figure 13: Altered Trafficking and Turnover in Neuraminidase-Deficient Fibroblasts WT and neuraminidase-deficient (ND) human fibroblasts were labeled with 50µM ManNAz, pulsed with Cmpd #188 and then chased for 0 hrs. (t 0 left column), 1 hr. (middle column), and 4 hrs. (right column). Neuraminidase is a hydrolase that cleaves the glycosidic linkages of sialic acid. Because ManNAz labels sialic acid residues in glycoconjugates, this experiment involving ND cells allowed us to view differences between correct and defective sialic acid trafficking and turnover within the cell. Note the cell-surface labeling in both the WT and ND t 0 samples. By 4 hours, the labeled ManNAz was properly turned over in the WT cells (after being sent to the Golgi). However, most still remains in the lysosomes of ND cells (see arrows). 23
CHAPTER 6 CONCLUSIONS Through the results from experiments performed involving cells affected by two types of storage disorders (Mucolipidosis-II and neuraminidase deficiency), it can be seen that the novel technique of click chemistry could be important in the study of trafficking and protein turnover, and cellular dynamics as a whole, in the field of glycobiology. The simplicity of using click chemistry makes tracking glycoproteins easier than previous techniques employed in the past, thus allowing for a suitable alternative for qualitative fluourescent evaluation. The azido-linked sugar analog ManNAz has proven to be useful in documenting the chemically-induced storage via the drug chloroquine (Figure 7); the compound also provided additional support for the lysosomal storage associated with ML-II (Figure 8). In addition to the important results associated with ML-II, the click chemistry also allowed for the study of intracellular trafficking (Figure 13). Gaining insight into the localization, trafficking and turnover of glycoproteins within cells isolated from lysosomal storage disorder patients may provide new insight into the pathophysiology of these diseases. We have demonstrated that azide sugar labeling and click chemistry can be an effective means of tagging these glycoproteins and visualizing their subcellular localization, intracellular accumulation and extracellular turnover. Since our findings are based on the use of only one azide sugar (ManNAz) and two different storage disorders, it will be interesting to expand this study in the future by utilizing different azide sugars (such as N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz) and 6-azidofucose (6AzFuc) to fluorescently tag O-linked, O-GlcNAc, and fucosylated glycoproteins, respectively) 24
and investigating other glycosylation disorders such as Congenital Disorders of Glycosylation or CDGs. Since CDGs involve defects in the biosynthesis of glycoproteins, as opposed to their breakdown or turnover, azide sugar techniques can be applied to determine subtle changes in the glycosylation state of cell surface glycoproteins. One intriguing observation of this study was the fact that extracellular matrix proteins were robustly visualized following ManNAz labeling. Most other studies that utilized azide sugar labeling employed cells grown in suspension and therefore lacked the production of an extracellular matrix environment. In our study, labeled extracellular matrix proteins were easily identifiable and stable. Indeed, long chase times were generally required to visualize turnover of these proteins. Since many of the antibodies for human matrix proteins are likely not crossreactive with other species such as feline or canine, the use of azide sugar labeling and click chemistry may provide a convenient means of tagging the extracellular environment in cultured primary cells from these important animal models. And although our primary readout in this study was fluorescence microscopy, tagging glycoproteins with azide sugars can also be used to isolate these proteins for analysis by Western blotting or mass spectrometry. This approach would be useful to determine whether specific glycoproteins are internalized more rapidly from the cell surface in diseased cells and should allow for a more quantitative assessment of the observed changes. The azido handle itself could also be incorporated into an array of intracellular macromolecules for tracking. Because the three-atom N 3 compound is mostly inert within the living environment and will not cause harm to the cells, it is not unreasonable to assume that the click chemistry could be used in compounds other than sugars, therefore, exploiting the powerful click chemistry technique. This technique could truly prove to be useful in the study of many 25
diseases, not just those with defective glycosylation. In addition to using click chemistry in cultured cells, it would be interesting to test the technique in living model organisms such as the zebrafish. The click chemistry has been used in zebrafish, 8 and the use of this technique could be the next step in determining some of the underlying problems that cause the defects seen in disease-affected organisms. Indeed, it seems that the possibilities of this exciting new revelation in glycobiology and pathology are endless. 26
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