1. N- Glycosylation, intracellular transport and sorting of recombinant bovine lactoferrin in polar and non- polar epithelial cells by Abdel-Salam, A. M., Abd El Gawad, I. A.*, El-Sayed, E. M.*, Mahfouz, M. B. and Naim, H**. Department of Food Technology and Dairying, National Research Centre,Cairo, Egypt. * Department of Dairy Science, Faculty of Agriculture, Cairo University, Giza, Egypt. **Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Germany.

2. Summary Recombinant bovine lactoferrin expressed in Cos-1 was treated with endo H and endo F, in order to demonstrate the type of glycosylation of these lactoferrin. The indicate that the lactoferrin in the cell was more mannose-rich protein and few complex form. With the lactoferrin secreted in the medium, molecular weight of the protein was almost unchanged when subjected to endo H hydrolysis, but gave a protein with less molecular weight when treated with endo F. This indicate the complex nature of the glycosylation in the lactoferrin secreted in the medium. Results clearly indicate that the mannose-rich species of lactoferrin was formed in the cell after 1 hour of pulse Chasing the formed lactoferrin indicated that lactofrrin was processed in the Golgi apparatus after 1-2 hour and the complex glycosylated lactoferrin was apparent in the medium within 2 hours of chasing.The sorting of bovine lactoferrin was carried out by biosynthetic labelling of polarized cells (MDCK cells) and the immunoprecipitation in the apical and basolateral medium and cell lysate. Results indicated that the transport of bovine lactoferrin (Lf) from trans-Golgi network to the apical surface. The role of N-glycosylation on the sorting of lactoferrin carried out by the culturing of the labeled MDCK cells in the presence or absence of glycosidase inhibitors.The resulted data indicate that impaired N-glycosylation may be played an important role in the transport of recombinant bovine lactoferrin in the presence of the glycosidase inhibitors.

3. INTRODUCTION Proteins are also glycosylated while in the ER lumen. Glycosylation is the addition of carbohydrate side chains to a protein. Proteins to which carbohydrates have been added are called glycoproteins. Carbohydrate moieties are quite important as recognition sites in cell-cell interactions, and in the targeting of proteins to the appropriate cellular compartment. Glycoproteins are classified as N-linked or O-linked, depending on the site of attachment of the carbohydrate chain. N-linked glycoproteins have carbohydrates, beginning with an N-acetylglucosamine, attached to the side chain of an asparagine residue. O-linked glycoproteins possess a carbohydrate link, beginning with an N-acetylgalactosamine, with a serine or threonine residue. Glycan moieties in Lf are thought to be involved in the functions such as anti-microbial activities and the protection of protein from proteolytic attack. Therefore, it is important to clarify the structure of glycan moieties in bLF to determine the relationship between the function and structure. The intracellular transport kinetics of bovine Lactoferrin in epithelial cells and the protein sorting were studied using the envelope glycoproteins of recombinant bovine lactoferrin as model apical and basolateral membrane proteins. This unique cellular organisation enables epithelia to carry out their specialized vectorial functions in secretion, absorption and ion transport.

4. MATERIALS AND METHODS I. Determination of the glycosylation of proteins Endo ß -N- acetylglucosaminidase H (endo H) and N-glycosidase F were used for detection of glycosylation of the obtained proteins.The two samples with enzymes were incubated at 37°C for 2-4 hours. When incubating under denaturating conditions overnight. The two incubated and control samples were mixed 30 µ l of Laemmli sample buffer and denatured at 95°C for 10 minutes. The samples were analyzed by SDS-PAGE and fluorography II. Intracellular transport kinetics of bovine Lactoferrin in epithelial cells Intracellular transport kinetics of bovine Lactoferrin in mammalian was carried out using pulse-chase experiments. Cells were washed two time with 5 ml mM HEPES. Prepared DNA solution (1.5 ml DMEM media +HEPES, 5 µ g DNA and 9 µ l of 50 mg /ml DEAE-dextran) was mixed and added to the washed cells. Labelling was performed for 1 h at 37  C followed by a chase with non-labelled methionine for different periods of time. The labelled cells were rinsed two times with phosphate buffered saline after medium collections. Cells were solublized with 1ml/dish of cold lysis buffer with protease inhibitor. III. N-gylcosylation and its roles in lactoferrin sorting to the apical and basolateral Membrane Immunoprecipitation – Metabolic labelling of MDCK cells grown on filters or plated in six-well culture dishes and MDCK clones expressing bovine lactoferrin were labelled for 1 h with 100 µ Ci [ 35 S]methionine and with glycosidase inhibitor agent (tunicmycine, Deoyxymannjirimycin (dMM),Deoyxynojirimycin (dNM) and swainsonine. Cell lysates and medium were immunoprecipitated from filters by addition of anti bovine lactoferrin to either the apical or basolateral compartments. The immunoprecipitates were analysed by SDS-PAGE.

5. RESULTS & DISCUSSION I. N-Glycosylation studies:To deterimine whether the recombinant lactoferrin was glycosylated in the same way as the natural lactoferrin, recombinant lactoferrin was treated with end H and N-glycosidase F. in order to demonstrate the type of glycosylation of these lactoferrin. Both enzymes has different specificities with respect to the linkage attached. endo H - + - - + - endo F - - + - - + LF-Lysate LF-Medium 116 97 66 49 29 Endo H hydrolyses at the glycosylamine linkage of the high- monnoseoligosaccharides form generating a carbohydrate free peptide of smaller molecular weight isolated from the cell lysate but can ’ t affect on the glycosylamine linkage in the complex oligosaccharides type, while N-glycosidase F hydrolyses at the glycosylamine linkage int the high- monnoseoligosaccharides and complex oligosaccharides form generating a carbohydrate free peptide of smaller molecular weight. Fig. (1) shows the SDS-PAGE of cell lysate and the medium after treatment with endo H and N glycosidase F. When lactoferrin from the cell lysate was treated with endo H and N-glycosidase F, the protein was affected more by the endo F hydrolase as a protein band of smaller molecular weight was apparent in the electrophoretic pattern while the lactoferrin was not affect much by by the endo H. This indicated that the lactoferrin in the cell was more mannose-rich protein and few complex form. With the lactoferrin secreted in the medium, molecular weight of the protein was almost unchanged when subjected to endo H hydrolysis, but gave a protein with less molecular weight when treated with N -glycosidase F. This indicate the complex nature of the glycosylation in the lactoferrin secreted in the medium

6. II. Intracellular transport kinetics of bovine Lactoferrin: Comparing the transport kinetics of lactoferrin by applying a pulse-chase protocol. Results (Fig. 2 ), clearly indicated that the mannose-rich species of lactoferrin was formed in the cell after 1 hour of pulse ( Culturing in medium containing S 35 labeled methionine). Chasing the formed lactoferrin indicated that the wild type of lactofrrin was processed in the Golgi apparatus after 1-2 hour and the complex glycosylated lactoferrin was apparent in the medium within 2 hours of chasing. This was in agreement with the published findings of the general procedure of membrane and secretory protein transport in the epithelial cells. The synthesized protein go to the ER and passed to Golgi apparatus to be in a complex form and secreted to medium. Chase (hour) 0.5 1 2 3 4 6 L M L M L M L M L M L M L: Cell Lysate & M: Medium Fig.(2 ): Intracellular transport kinetics of bovine Lactoferrin in Cos-1 cells time from 0.5 until 6 hours

7. Lysate Meduim apical basolateral III. Lactoferrin sorting in the epithelial cells and its relation to the N-glycosylation The simple epithelia, lining the body cavities, form cellular sheets between the external and internal environment. In order to perform their boundary function, epithelial cells have evolved a very specialized cellular architecture. Their membrane is divided into two domains with distinct protein and lipid compositions. An apical domain confronts the outside world and a basolateral domain which is surrounded by the body fluids and promotes attachment to the extra cellular matrix. The Madin-Darby canine kidney (MDCK) cell system are used as experimental model. MDCK cells were cultured on filter and medium were added up (Apical medium) and down (basolateral medium). The biosynthetic labelling of MDCK cells were carried out using S 35 methionine and the immunoprecipitation in the apical and basolateral medium and cell lysate. Results indicated that the intracellular transport and sorting of bovine lactoferrin (Lf) in epithelial cells was to the apical membrane Fig. (3 ) Fig. (3): The sorting of proteins and transport to the apical and basolateral membranes in MDCK cells apical membrane basolateral membrane Golgi ER Endosomes TGN Sorting Signals Apical membrane GPI N-glycans? O-glycans? Proteinaceous patches? Transmembrane domains? Cytoplasmic peptides? Basolateral membrane Cytoplasmic peptides YXX , LL or IL

8. In order to study the role of N-glycosylation on the intracellular transport of lactoferrin labeled MDCK cells were cultured in the presence or absence of glycosidase inhibitors. MDCK cells line expressing bovine lactoferrin were labeled with ( S 35 ) methionine and then cultured in the following glycosidase inhibitors: 4 µ g/ml tunicmycine, 5mM deoxymannjirimycin (dMM), 5mM deoxynojirimycin (dNM), 5mM Swainsonine and 5mM Benzyl. When Tunicmycin was added in the medium a dramatic decrease in the molecular weight of the cell lysate, lactoferrin was apearent unclear sorting and slowly transport to the apical or basolateral membrane. Also, in the presence of the another glycosidase inhibitors, lactoferrin sorting was unclear and lactoferrin transport was slowly and the lactoferrin was concentrated in the cell lysate Fig. (4). Control Tunicmycin DMM DNM Swansionin Benzyl L A B L A B L A B L A B L A B L A B L: Lysate A: Apical medium B: Basolateral medium membrane Fig. (4): Lactoferrin sorting in the presence and absence of glycosidase inhibitors. The resulted data indicate that impaired N-glycosylation may be played an important role effects on the transport kinetics of lactoferrin in the presence of the glycosidase inhibitors.

9. CONCLUSION N-glycosylation studies indicated that the recombinant bovine lactoferrin in the cell was more mannose-rich protein and few complex form and the complex nature of the glycosylation in the lactoferrin secreted in the medium. The transport kinetics of rbLf clearly indicated that the mannose-rich species of lactoferrin was formed in the cell after 1 hour of pulse and chasing the formed lactoferrin indicated that the wild type of lactofrrin was processed in the Golgi apparatus after 1-2 hour and the complex glycosylated lactoferrin was apparent in the medium within 2 hours of chasing. The sorting studies of rbLf indicated that the intracellular transport and sorting in epithelial cells was to the apical membrane. The role of N-glycosylation on the sorting of rbLf in the presence or absence of glycosidase inhibitors indicated that impaired N-glycosylation may be played an important role in the transport kinetics of lactoferrin in the presence of the glycosidase inhibitors.