Teaching Glycoproteins with a Classical Paper: Knowledge and Methods in the Course of an Exciting Discovery

Transcription

1 Q 2008 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 36, No. 5, pp , 2008 Articles Teaching Glycoproteins with a Classical Paper: Knowledge and Methods in the Course of an Exciting Discovery THE STORY OF DISCOVERING HK-ATPASE b-subunit Received for publication, March 21, 2008, and in revised form, May 14, 2008 Lixin Zhu From the Department of Molecular and Cell Biology, University of California, Berkeley, California To integrate research into the teaching of glycoproteins, the story of discovering hydrogen-potassium ATPase (HK-ATPase) b subunit is presented in a way covering all the important teaching points. The interaction between the HK-ATPase a subunit and a glycoprotein of kda was demonstrated to support the existence of the b subunit. On revealing the strategies and experimental designs of this discovery, the knowledge of glycoproteins is delivered. The purpose of this effort was to facilitate the teaching of scientific thinking in the science classroom, to make the biochemistry classroom a more interesting place, and to attract uncertain minds into the career of biochemistry research. Keywords: Glycoprotein, lectin, glycosidase, hydrogen-potassium ATPase. To whom correspondence should be addressed. Department of Molecular and Cell Biology, 245 LSA, MC #3200, University of California, Berkeley, CA , USA. Tel.: ; Fax: ; zhulx@berkeley.edu. 1 The abbreviations used are: HK-ATPase, hydrogen-potassium ATPase; NaK-ATPase, sodium-potassium ATPase; horseradish peroxidase, HRP; DTAB, dodecyltrimethylammonium bromide. DOI /bmb Beautiful facts, harmonious relationships between structures and functions, and elegant mechanisms at the molecular level are rapidly being discovered and presented in biochemistry literatures and squeezed into already crowded biochemistry textbooks. The vast amount of knowledge in all sections of the chemistry of life leaves little time in biochemistry lecture for anything else. After gaining all the necessary knowledge from biochemistry classes, undergraduate students would not necessarily know what biochemistry looks like in practice, how biochemical research is done, or how exciting discoveries are made. A way to balance the teaching of biochemical theory and the teaching of biochemistry research is to integrate research article(s) into undergraduate teaching [1]. The lecture can be made more interesting to students if they see that the textbook knowledge was integrated into experiments that lead to important discoveries. This makes the material more memorizable. In addition, students will gain a sense for exciting and cutting-edge research. The longterm effect of this effort is to attract some uncertain minds into the career of biochemistry research. I luckily came across a classical paper [2] describing the discovery of the b-subunit of the gastric hydrogenpotassium ATPase (HK-ATPase), 1 the proton pump responsible for the secretion of gastric acid, which kills most of the germs in food and activates pepsinogen for protein digestion in the stomach. When this 1990 article was written by Dr. Okamoto and his colleagues in the laboratory of Dr. John G. Forte at the University of California, Berkeley, the contents presented in the biochemistry textbooks on glycoproteins were scarce. However, the authors/investigators were familiar with recent progresses in glycoprotein research and they put them into practice leading to the exciting discovery of the HK-ATPase holoenzyme. Much of the glycoprotein knowledge applied in this research appears in the current syllabus of undergraduate biochemistry. Thus, it is amazingly possible to exhaust all the teaching points of glycoprotein with mainly one research article. Here, this article of milestone value in the field of gastroenterology is presented again to convey the knowledge of glycoproteins to the undergraduates. More importantly, the secret strategies and experimental designs of biochemical discovery from this article are revealed to the curious minds of future biochemists. The design of this module is to link all the teaching points on glycoproteins together within a clear, simplified story of discovery, which is in stark contrast to a plain, conventional delivery of the knowledge. Research articles with simple logic and clear results are desirable tools for this teaching method because the major function of this module is to reinforce the learning of the basic knowledge of biochemistry. In an introductory course, one would not have the luxury of spending 30 min dissecting the structure of a dense research article. The article presented here happens to be such a lucid (albeit long) article. In addition, the article is special in that most of the This paper is available on line at

2 337 basic knowledge of glycoprotein for an undergraduate classroom is an underlying element of the research. Meanwhile, the discovery is important to our daily life (actually, every time we eat). With not much more material included than a conventional biochemical lecture, this module is designed to fit in a 50-min lecture in any given size of classroom. As a replacement for a conventional lecture in an introductory biochemistry, the preliminary knowledge required would also remain the conventional knowledge of basic general biology or organic chemistry. Accordingly, the assessment of the teaching effectiveness is the same as that of a conventional biochemistry course. Assuming that the same teaching points including biochemical facts and methods are to be covered, the advantage of teaching with this article is expected to help students understand how to discover a new component in a protein complex and how to study protein protein interactions. BACKGROUND In the very beginning of the lecture, the introductory information would be given about the various important functions of glycoproteins. Depending on the familiarity of the instructor in different fields, examples can be chosen from: 1) structural, organizational, and barrier functions (e.g. mucins on the gastric epithelium); 2) immune regulation (e.g. the heavily glycosylated envelope protein E2 of hepatitis C virus enable this virus to evade the antiviral effects of IFN); 3) cell signaling (many hormones, growth factors, and the receptors for these factors are glycoproteins, e.g. b-hcg, erythropoietin, insulin receptor); 4) cell-to-cell adhesion and contact inhibition (e.g. binding of lymphocytes to selectins on certain inflammatory epithelium cells). The Band 3 glycoprotein in the ABO blood type system may be given as an example of how the knowledge of glycoproteins is important in medicine. Then the discovery story begins. The catalytic a subunit of HK-ATPase was discovered in 1967 by Forte et al. [3]. This peptide has all the binding sites for proton, potassium ion, and ATP. The catalytic domain for hydrolyzing ATP also resides in this peptide. It was thus considered the sole peptide of the proton pump until the late 1980s. The major mechanism for the regulation of the proton pump activity was also established prior to the discovery of the b subunit, in a 1977 article (by Dr. Forte together with another Dr. Forte and Dr. Machen, all from University of California, Berkeley) describing the discovery of the massive recycling of membranes between the apical membranes and intracellular tubular vesicles of the oxyntic cell (parietal cell) [4]. Thus, there seemed to be no need for another HK- ATPase subunit. The motivation to look for the other subunit came from two discoveries. One was the famous b subunit of sodium-potassium ATPase (NaK-ATPase), which is responsible for the maintenance of the membrane potential of many cells. The NaK-ATPase, HK-ATPase, and calcium ATPase are three members of the same protein family, FIG. 1.The schematic representation of the hydrogen-potassium ATPase (HK-ATPase) holoenzyme hypothesis. This model features with a b subunit carrying multiple sites of glycosylation, which covers much of the luminal side of the HK- ATPase. Accurate information regarding the N-glycosylation sites and the glycan structures can be found in [5, 6]. which share significant similarities in protein sequences, conformations, and activities. Researchers often look into proteins of the same family to gain novel ideas and insights. It was known that the NaK-ATPase, but not the calcium-atpase, has a b subunit carrying heavy glycosylation. From these two close relatives, it seemed that there was a 50% possibility that HK-ATPase would have a b subunit. The other discovery favoring a possible b subunit was the presence of glycoproteins in the intracellular tubular vesicles, which is the resting pool of HK- ATPase in parietal cells. This fact made the 50% chance shining. Thus the hypothesis came into existence (Fig. 1). With this cartoon drawing of HK-ATPase, it is time to describe the basic structures of glycoproteins, the N-linkage and the O-linkage of the sugar chains, and the orientation of the glycoproteins on plasma membrane. To explain why most of the glycosylation is on the extracellular side, the location for glycosylation modification can be presented. Asparagine (N-linked) and serine/threonine residues (O-linked) are glycosylated during passage through the endoplasmic reticulum and Golgi apparatus in eukaryotic systems. STRATEGY AND METHODS To test the hypothesis, the investigators needed to show a specific stable interaction between HK-ATPase and the suspected glycoprotein. To demonstrate a stable interaction between protein A (HK-ATPase) and B (a glycoprotein), biochemists usually take these two steps: 1) purify protein A without disrupting stable protein protein interactions, hoping to see protein B, but not other proteins, copurified; 2) purify protein B in a similar mild procedure, hoping to see protein A, but not other proteins, copurified. The first step was easy to achieve because coimmunoprecipitation could be performed with an anti-hk-atpase a subunit antibody. Methods are available to detect the copurified peptide and the presence of glycosylation on the peptide. The second step was hard because nothing was known about the b subunit except that it was putatively a glycoprotein. How would they purify something unknown? We see from the article that their strategy was to exploit the characteristic and specific interaction between glycoproteins and lectins, which was a newly established method when this study was carried

3 338 BAMBED, Vol. 36, No. 5, pp , 2008 out (Fig. 2). Lectins are sugar binding proteins that have a selective affinity for carbohydrate moieties. Now is the time to show students the different types of monosaccharides attached to glycoproteins and their specific interactions with different types of lectins, for examples, Con A, WGA. After extraction with the nonionic detergent NP-40, the solubilized membrane glycoprotein was fished out by lectins. The next exciting part of the experiment was to find out if the HK- ATPase alpha subunit was pulled down simultaneously, which was to be determined by western blot analysis using an anti- HK-ATPase a subunit antibody. FIG. 2. Examples of widely used lectins. Concanavalin A (Con A) binds to a-linked mannose, which presents in all N- linked glycans, whereas wheat germ agglutinin (WGA) binds to terminal N-acetyl-glucosamine (GlcNAc) and sialic acid (Sia) residues. Man, mannose; Gal, galactose. RESULTS Immunoprecipitation of HK-ATPase Alpha Simultaneously Brought Down a Glycoprotein Immunoprecipitation with an HK-ATPase a subunit antibody was able to enrich the HK-ATPase a subunit, and, at the same time, pulled down a broad protein band at kda in the SDS-PAGE gel. No other protein was detected in the immunoprecipitated product. What was this kda band(s)? This diffusive band on a protein gel is a typical characteristic of glycoprotein because the multiform glycosylation on the same protein could variably affect the mobility of the protein in the gel. First, this protein was characterized by Coomassie blue staining and quantitated by the Bradford method of protein assay. Detection of sugar chains in SDS-PAGE gel was done using the method of periodic acid-dansyl hydrazine staining. This staining method is based on periodic acid oxidation of a substance containing 1,2-glycol grouping. The resulting dialdehyde then reacts with a Schiff s base (e.g. dansyl FIG. 3. Specificities of some widely used glycosidases. Endoglycosidase F (Endo F) cleaves between the two GlcNAc residues in the N-linked oligosaccharide, generating a truncated sugar molecule with one GlcNAc residue remaining on the asparagine residue. High mannose (oligomannose) and hybrid structures can be removed by Endo F1, but not complex oligosaccharides. Endo F2 cleaves biantennary complex and, to a lesser extent, high mannose oligosaccharides. Endo F2 will not cleave hybrid structures. Endo F3 cleaves biantennary and triantennary complex structures, but not oligomannose or hybrid structures. PNGase F cleaves all asparagine-linked complex, hybrid, or high mannose oligosaccharides unless a(1 3) core fucosylated. Endoglycosidase H (Endo H) cleaves between the GlcNAc residues of the N-linked glycans. Oligomannose and most hybrid-type glycans, including core fucosylated, are hydrolyzed by Endo H. However, complex-type oligosaccharides are not hydrolyzed.

4 339 The lectin chromatography performed in the presence of DTAB was able to enrich the same five glycoproteins. But this time, HK-ATPase a subunit was not present in the eluant, indicating no glycosylation on a subunit. The above results together made it clear that the retention of HK-ATPasea subunit on the lectin column was because of its interaction with the gp60 80 (Fig. 5). FURTHER CHARACTERIZATION OF THE SUGAR CHAINS ON HK-ATPASE b SUBUNIT Without an introduction about mass spectrometry, the glycoprotein lecture is not going to be complete. If time FIG. 4. Determination of the stoichiometry between HK- ATPase a and b subunit. Oligosaccharides were removed to determine the relative amount of HK-ATPase b subunit because 1) variability of the glycosylation level on different molecules cause variability of molecular weight; 2) oligosaccharides have a negative effect on protein quantification by the method of Coomassie blue staining. After treating with Endo F, which removed most of the carbohydrates, the HK-ATPase subunits were separated in a gel and stained with Coomassie blue. The gel image was scanned and the integrated density values (IDV) were obtained for HK-ATPase a and b subunits. These values and the molecular mass of these two peptides were used to calculate the molar ratio (stoichiometry) between a and b subunits. hydrazine) to form a colored product. The fact that this is a glycoprotein(s) was also confirmed by western blot analysis using horseradish peroxidase (HRP)-conjugated lectin. To find out how many peptides were present in the kda broad band, the sample was treated with glycosidase (Endo F) followed by SDS-PAGE electrophoresis. The glycosidases of different specificity are shown to the students at this time so that they understand that the investigators were using an enzyme capable of removing most of the N-linked sugars from the glycoprotein(s) (Fig. 3). Only one protein band showed up after deglycosylation of the glycoprotein kda (gp60 80). The deglycosylated sample also made it possible to determine the stoichiometry of the HK-ATPase a subunit to the proposed b subunit (gp60 80), which is about one (Fig. 4). HK-ATPase a Subunit was Copurified by Lectin Chromatography Lectin chromatography enriched five glycoproteins, with the gp60 80 being the dominant one, and one extra strong band at about 110 kda, which is the HK-ATPase a subunit as revealed by western blot analysis. With these results, can we draw the conclusion that HK-ATPase alpha was copurified because it binds to the gp60 80, which was trapped by lectin? Not yet. The question is that could HK-ATPase a subunit have been purified because the a subunit itself is a glycoprotein, as claimed in an earlier article [7]? To clear this concern, the investigators created a perfect situation where protein protein interaction was abrogated while lectin carbohydrate interaction remained by using a denaturing cationic detergent dodecyltrimethylammonium bromide (DTAB), which is another powerful tool in glycoprotein research. FIG. 5.Dodecyltrimethylammonium bromide (DTAB) experiments suggested the interaction between a and b subunits of HK-ATPase. (a) Lectin chromatography in the presence of mild detergent which do not disrupt protein protein interactions. In this experiment, both HK-ATPase a and bsubunits were retained by the lectin column. (b) Lectin chromatography in the presence of cationic detergent DTAB, which disrupts the interaction between a and b subunits of HK-ATPase. Thus, only the glycosylated b subunit was retained by the lectin column. (c) If a subunit is a glycosylated peptide, it should also bind to the lectin column in the presence of DTAB, which did not happen.

5 340 BAMBED, Vol. 36, No. 5, pp , 2008 TABLE I Glycoprotein teaching points covered in the Okamoto article Facts 1 Basic structures, N-linkage, and O-linkage 2 Orientation of glycoprotein on plasma membrane 3 Location of glycosylation modification 4 Different types of monosaccharides and their binding to lectins 5 Glycosidases of different specificity Methods 1 Lectin affinity chromatography 2 Glycoprotein staining method with periodic acid-schiff s base 3 Western blot analysis using HRP-conjugated lectin 4 Mass spectrometry in deciphering the glycans structures allows, the principle of mass spectrometry may be explained briefly. Dr. Forte and his colleagues used mass spectrometry, in conjunction with glycosidase digestion, Edman degradation, and monosaccharide composition analysis, to analyze the glycosylation sites and the structures of the N-linked oligosaccharides of the HK-ATPase b subunit [5, 6]. This is a good example of how mass spectrometry helps to reveal the glycan structures on glycoproteins. WHAT IS THE IMPORTANT FUNCTION OF HK-ATPASE b SUBUNIT? Because all the binding and catalytic domains are located on the a subunit, is the b subunit of HK-ATPase just a pretty decoration? Of course it is not. HK-ATPase b knockout mice were made in Dr. van Driel s lab in Australia [8]. These knockout mice could not secret HCl into the stomach lumen. The consequence of b subunit knockout is thus similar to the consequence of knocking out the a subunit of HK-ATPase [9]. However, the molecular mechanism of the b subunit function is not clear and currently being investigated. CONCLUSIONS The story of exciting discoveries will never end. But the lecture can be paused here. In this lecture, all the important teaching points (Table I) of glycoprotein can be covered while presenting the Okamoto article. In addition, the strategy for identification of a new component of a protein complex or the detection of protein protein interaction can be disclosed. Generally, students find this sort of presentation useful. Specifically, they think it is interesting to learn about basic methodology in the context of a research story while learning facts about glycoproteins. They also think that it is a definite possibility that students who are not sure what field they are interested in may be attracted by the excitement shown in novel discovery. However, they raised an interesting argument that the opposite could occur as well: borderline students considering research as a career or extracurricular activity as undergrads may be dissuaded by the stronger methodology results linkage presented here and may decide not to pursue that avenue. And either possibility is considered a benefit: helping borderline students decide as early as possible is an advantage that saves both student and teacher/ mentor in the lab time and energy. This manner of presentation is conducive to help students decide what to do with their futures, rather than just presenting them with facts but without the type of work that they could become involved in to discover similar facts. Acknowledgments The author thanks Kevin Poon, Jason Hatakeyama, and Bing Zhang (University of California, Berkeley) for their critical reading of the manuscript and for their valuable suggestions/feedbacks. REFERENCES [1] S. Schuster (2006) Commentary: The business of the biosciences can be integrated into a biochemistry curriculum, Biochem. Mol. Biol. Educ. 34, [2] C. T. Okamoto, J. M. Karpilow, A. Smolka, J. G. Forte (1990) Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated beta-subunit of the Hþ/K(þ)-ATPase, Biochim. Biophys. Acta 1037, [3] J. G. Forte, G. M. Forte, P. Saltman (1967) Kþ-stimulated phosphatase of microsomes from gastric mucosa, J. Cell Physiol. 69, [4] T. M. Forte, T. E. Machen, J. G. Forte (1977) Ultrastructural changes in oxyntic cells associated with secretory function: A membranerecycling hypothesis, Gastroenterology 73, [5] K. Tyagarajan, P. H. Lipniunas, R. R. Townsend, J. G. Forte (1997) The N-linked oligosaccharides of the beta-subunit of rabbit gastric H,K-ATPase: Site localization and identification of novel structures, Biochemistry 36, [6] K. Tyagarajan, R. R. Townsend, J. G. Forte (1996) The beta-subunit of the rabbit H,K-ATPase: a glycoprotein with all terminal lactosamine units capped with alpha-linked galactose residues, Biochemistry 35, [7] W. H. Peters, A. M. Fleuren-Jakobs, J. J. Schrijen, J. J. De Pont, S. L. Bonting (1982) Studies on (Kþ þ Hþ)-ATPase V. Chemical composition and molecular weight of the catalytic subunit, Biochim. Biophys. Acta 690, [8] K. L. Scarff, L. M. Judd, B. H. Toh, P. A. Gleeson, I. R. Van Driel (1999) Gastric H(þ),K(þ)-adenosine triphosphatase beta subunit is required for normal function, development, and membrane structure of mouse parietal cells, Gastroenterology 117, [9] Z. Spicer, M. L. Miller, A. Andringa, T. M. Riddle, J. J. Duffy, T. Doetschman, G. E. Shull (2000) Stomachs of mice lacking the gastric H,K-ATPase alpha-subunit have achlorhydria, abnormal parietal cells, and ciliated metaplasia, J. Biol. Chem. 275,