©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Mutational Analysis of the Glycosylation Sites of Human Keratin 18 (*)

(Received for publication, January 13, 1995; and in revised form, March 20, 1995 )

Nam-On Ku (§) M. Bishr Omary (¶)

From the  Palo Alto Veterans Administration Medical Center, Palo Alto, California 94304 and the Digestive Disease Center, Stanford University School of Medicine, Stanford, California 94305

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament phosphoglycoproteins that are expressed preferentially in glandular epithelia. We previously showed that K8/18 phosphorylation occurs on serine residues and that K8/18 glycosylation consists of O-linked single N-acetylglucosamines (O-GlcNAc) that are linked to Ser/Thr. Since the function of these modifications is unknown, we sought as a first step to identify the precise modification sites and asked if they play a role in keratin filament assembly. For this, we generated a panel of K18 Ser and Thr Ala mutants at potential glycosylation sites followed by expression in a baculovirus-insect cell system. We identified the major glycosylation sites of K18 by comparing the tryptic ^3H-glycopeptide pattern of the panel of mutant and wild type K18 expressed in the insect cells with the glycopeptides of K18 in human colonic cells. The identified sites occur on three serines in the head domain of K18. The precise modified residues in human cells were verified using Edman degradation and confirmed further by the lack of glycosylation of a K18 construct that was mutated at the molecularly identified sites then transfected into NIH-3T3 cells. Partial or total K18 glycosylation mutants transfected into mammalian cells manifested nondistinguishable filament assembly to cells transfected with wild type K8/18. Our results show that K18 glycosylation sites share some features with other already identified O-GlcNAc sites and may together help predict glycosylation sites of other intermediate filament proteins.


INTRODUCTION

Keratin intermediate filaments (IF),^1(^1) which are preferentially expressed in epithelial cells (1, 2) , undergo several post-translational modifications including phosphorylation (3-7) and glycosylation (8, 9, 10, 11, 12, 13) . Cells in different epithelia express varying complements of keratins that form obligate heteropolymers in a cell-specific manner. For example, glandular type ``simple'' epithelia typically express keratin polypeptides 8 and 18 (K8/18) (1) . In general, the function of IF proteins is poorly understood, although at least one function for epidermal keratins is to provide cells with structural stability (reviewed in Refs. 14-17). The most striking evidence for this is the identification of several inherited blistering skin diseases that are caused by mutations in epidermal keratins (reviewed in Refs. 18-20). In the case of K8/18, there is no clear function or human disease association, but gene disruption of mouse K8 resulted in fetal death with extensive liver hemorrhage (21) or in colorectal hyperplasia and rectal prolapse (22) depending on the genetic background of the mice. If one accepts the hypothesis that post-translational modifications of IF proteins are likely to regulate their function(s), then characterization of these modifications should provide a handle for studying the function and regulation of these proteins.

With regard to IF phosphorylation, there are accumulating data that this modification plays an important role in filament reorganization (reviewed in Refs. 23 and 24). In contrast with IF phosphorylation, IF glycosylation is a more recently recognized modification that has been well characterized on K13 (10) , K8/18 (11) , and neurofilaments (25) . IF glycosylation consists of single N-acetylglucosamine (GlcNAc) residues that are O-linked to serine/threonine (Ser/Thr). This modification was first described in mouse lymphocytes (26), and, since then has been shown to be broadly distributed in many nuclear and cytoplasmic proteins (reviewed in Refs. 27 and 28). The function of this modification is unknown, but all proteins identified with this modification share the feature of forming multimeric complexes (27, 28) . This suggests that this modification plays a potential role in protein-protein interaction. The likely importance of the O-GlcNAc modification is its dynamic nature (11, 29) , which together with its simplicity and potential attachment sites (Ser/Thr) make it similar to phosphorylation (27, 28) . Interestingly, the K8 and K18 molecules that are phosphorylated are for the most part not glycosylated and vice versa, which suggests that each modification may have different regulatory functions or that one modification may block or regulate occurrence of the other (12) . However, in the case of K8/18, several lines of evidence suggest that the latter possibility is unlikely (7) .

In this study, we used a mutational approach coupled with manual Edman degradation to identify the major glycosylation sites of human K18 and showed that mutation of these sites does not appear to play a significant role in filament assembly in transfected cells. Our study is based on the following earlier data. (i) K18 glycosylation occurs within the N-terminal 125-amino acid domain which ends with a biochemically cleavable tryptophan (Fig. 1) (12) . (ii) Human K8/18 can be efficiently expressed in insect Sf9 cells, using baculovirus recombinants, to allow for detailed biochemical studies. In addition, K18 displays highly conserved glycosylation in the insect and human cell systems (30) . (iii) We have successfully used a similar mutational approach to identify the major phosphorylation site of human K18 (7) .


Figure 1: Amino acid sequence and tryptic peptides of K18 head and proximal rod domains. Single-letter abbreviations are used to show the amino acid sequence of human K18 beginning with the acetylated serine. Brackets enclose predicted trypsin digestion products. Serine residues are shown in bold italics and are numbered consecutively above the brackets, with numbers below indicating the amino acid position. The beginning of the rod domain and end of the head domain are also shown. Asterisks highlight the eight threonine residues.




MATERIALS AND METHODS

Cells and Reagents

The cell lines used were: HT29 (human colon), NIH-3T3 (mouse fibroblast), and BHK-21 (hamster kidney). They were obtained from the American Type Culture Collection (Rockville, MD) and cultured as recommended by the supplier. Insect Sf9 cells were from Pharmingen (San Diego, CA). Monoclonal antibody L2A1, which recognizes human K18, was used for immunopurification of K8/18 (11) . Other reagents used were: uridine diphosphate (UDP)-[4,5-^3H]galactose (36.7 Ci/mmol) and EN^3HANCE spray (DuPont NEN), galactosyltransferase (Sigma), and trypsin (Worthington Biochemical Corp.).

Construction of Mutants and Baculovirus Recombinants

The cDNA for K8 and K18 were subcloned into the pBluescript SK plasmid (7) . Site-directed mutagenesis to generate serine and threonine alanine (Ala) mutants were carried out using a Transformer kit (Clontech). For mutations at adjacent serines or threonines, a single primer was typically used, whereas multiple primers were used for mutations at nonadjacent residues. All mutations were confirmed by sequencing the mutagenized regions. Mutants were subcloned into the pVL1392 vector for expression in Sf9 cells (30) or downstream of the hCMV promoter in the pMRB101 mammalian expression vector (31) . Baculovirus recombinants were generated using the BaculoGold Transfection kit (Pharmingen) exactly as described (30).

Immunofluorescence Microscopy

Transfected mammalian cells were grown on coverslips using 6-well plates. After 3 days of transient transfection, cells were fixed for 3 min in -20 °C methanol and then washed. Cells were then incubated with monoclonal antibody L2A1 (30 min), washed, and then incubated with Texas Red-conjugated goat anti-mouse antibody (30 min). Color slides were taken using Ektochrome Kodak Elite 400 film from which black and white pictures were generated.

Immunoprecipitation and Tryptic Peptide Mapping

Immunoprecipitation was carried out using Sf9 cells infected with the recombinant baculovirus constructs (4 days) or NIH-3T3 cells transfected with the mammalian expression vector constructs (3 days). Cells were solubilized with 2% Empigen BB in phosphate-buffered saline containing 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 25 µg/ml aprotinin, 10 µM leupeptin, 10 µM pepstatin (45 min, 4 °C) (32) . The nonsolubilized residual was pelleted, followed by incubation of the solubilized material with monoclonal antibody L2A1 conjugated to agarose (1-2 h), and then washing. Galactosylation of the K8/18 immunoprecipitates was carried out using UDP-[^3H]galactose and galactosyltransferase exactly as described (12) .

Tryptic glycopeptide mapping was done using labeled K18 which was isolated from K8/18 immunoprecipitates using preparative SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The K18 bands were visualized by brief Coomassie Blue staining, followed by electroelution and then acetone precipitation. Keratin precipitates were digested with trypsin (tosylphenylalanyl chloromethyl ketone-treated) (375 µg/ml in 80 µl of 50 mM NH(4)HCO(3)) for 16-24 h, followed by lyophilization, then analysis in the horizontal dimension by electrophoresis (pH 1.9 buffer) and in the vertical dimension by chromatography using cellulose glass plates (10,000-15,000 cpm loaded per plate) as described (33) . After being allowed to air-dry, plates were evenly sprayed with EN^3HANCE and then exposed for 7-14 days.

Manual Edman Degradation

Individual K18 glycopeptides, isolated from HT29 or Sf9 cells, were water-extracted from their corresponding spots on the cellulose plates (after fluorography and using the developed films as templates) as described (33) . Prior to extraction, the plates were placed in a fume hood to allow evaporation of the EN^3HANCE material. The extracted peptides were lyophilized and then subjected to manual Edman degradation exactly as described (25) .

RESULTS

Localization of the Major Tryptic Glycopeptides of K18

Our previous results showed that human K18 glycosylation in HT29 colonic tumor cells occurs within the N-terminal 125-amino acid domain that is generated after tryptophan cleavage using HCl/dimethyl sulfoxide/HBr (Fig. 1) as described previously (12) . This domain (Fig. 1) contains 20 and 8 of the potential 37 serine and 30 threonine glycosylation sites of K18, respectively, based on the known primary structure of human K18 (34, 35) . Since initial attempts at obtaining unambiguous sequences after isolating radiolabeled glycopeptides followed by microsequencing were unsuccessful in our hands (not shown), we elected to use a molecular mutational approach to localize the tryptic peptides where K18 glycosylation occurs. To do so, we used a baculovirus expression system in insect Sf9 cells to generate large quantities of K18 proteins that were mutated at potential Ser/Thr K18 glycosylation sites. The basis for this approach is the conserved glycosylation when comparing human K18 isolated from HT29 cells or expressed in insect cells (30) . As described previously (30) , immunoprecipitation of K18 from HT29 cells or Sf9 cells, followed by labeling of accessible terminal GlcNAcs, showed nearly identical tryptic glycopeptides. The tryptic ^3H-glycopeptide profile of K18 (human or after expression in Sf9 cells) is exemplified by the profile shown in Fig. 2A, which shows that K18 contains three major tryptic glycopeptides. The other unnumbered spots in Fig. 2A are sometimes seen and correspond to labeled galactosyltransferase enzyme contamination (not shown and Ref. 30).


Figure 2: Characterization of the glycosylation of K18 serine mutants expressed in Sf9 cells. K8/18 immunoprecipitates were obtained from Sf9 insect cells infected with K8/18 recombinant baculovirus. The immunoprecipitates were labeled with UDP-[^3H]galactose using galactosyltransferase. Radiolabeled K18 was eluted from the gel, digested with trypsin, and then analyzed in the horizontal dimension by electrophoresis and in the vertical dimension by chromatography as described under ``Materials and Methods.'' A representative group of K18 serine constructs from Table I is shown with numbers corresponding to mutated serines indicated as consecutive numbers in Fig. 1. Numbered spots correspond to major labeled K18 glycopeptides. Note the absence of peptide 3 in B and peptides 1 and 2 in C and D.




Table I


In order to assign the three major tryptic glycopeptides shown in Fig. 2A to specific K18 tryptic peptides, we generated a series of K18 constructs that have single or multiple mutations at serine or threonine residues. These mutants were then expressed as recombinant baculoviruses in Sf9 cells followed by immunoprecipitation of K8/18, labeling of the K18 glycosylation sites, and then tryptic peptide mapping to determine which mutation results in the absence of labeling of the specific peptides. As shown in Table I, mutation of all the potential threonine glycosylation sites did not affect the labeling of any of the glycopeptides. Hence, all the threonine mutant constructs showed K18 tryptic glycopeptide maps that appeared identical (not shown) with that of the wild type K18 map shown in Fig. 2A. In contrast, mutation of some of the serine residues did abolish the glycosylation of specific tryptic peptides (Table I). For example, constructs that contained mutations of Ser-12,13, Ser-12-15, and Ser-4,5,9,12-15 showed lack of labeling of peptide 3 as represented by the map of Ser-12,13 shown in Fig. 2B. This suggests that glycosylation of peptide 3 occurs on Ser-12 and/or -13. Similarly, constructs that contained mutations of Ser-7-9 and Ser-7,8,11,14 showed lack of labeling of peptides 1 and 2 as shown in the maps of Fig. 2D and Fig. 2C, respectively. This suggests that glycosylation of peptides 1 and 2 occurs on Ser-7 and/or -8. Confirmation of the assignment of the peptide numbers shown in Fig. 2, B-D, was obtained by performing mixture maps with the wild type construct (not shown). The four minor spots seen in Fig. 2 , C and D, are background spots that are also seen in A and B of Fig. 2. Of note, the construct containing mutations of Ser-7-9 resulted in the disappearance of spots 1 and 2 (Fig. 2D) even though the mutations are contained within the same predicted tryptic peptide (see Fig. 1 ). This may be consistent with incomplete trypsin digestion at Arg-26 or the presence of two forms of the Ser-7-11-containing peptide (e.g. phosphorylated or other post-translationally modified forms), or that mutations of Ser-7-9 indirectly alter the glycosylation at distant sites of other tryptic peptides (see below).

Identification of the Major Glycosylation Sites of Human K18

The results shown in Table Iand Fig. 2indicated that one or both serines in the Ser-12,13- and the Ser-7,8-containing peptides are glycosylated. In order to identify the precise modification residues, we generated individual and multiple serine mutants of K18 that cover these potential glycosylation sites. As shown in Fig. 3A, expression of the mutants in Sf9 cells using recombinant baculoviruses resulted in significant protein levels as noted after immunoprecipitation and Coomassie staining of the SDS-PAGE separated products. Analysis of the glycosylation of the individual serine K18 mutants: Ser-7, Ser-8, Ser-12, and Ser-13 using one-dimensional SDS-PAGE was unrevealing except that some decrease in the glycosylation of the Ser-8 as compared with the wild type (WT) K18 construct was observed (Fig. 3A, lanes a-e). However, analysis of the tryptic glycopeptide maps of the individual serine mutants showed that mutation of Ser-13 (i.e. Ser-48 of K18, Fig. 1) abolished glycopeptide 3 (Fig. 3B, compare panels a and b). Furthermore, although individual Ser-7 and Ser-8 constructs showed tryptic glycopeptide maps that were identical with wild type K18 (not shown), the double mutant construct Ser-7,8 (Fig. 3A, lane f) showed the absence of glycopeptides 1 and 2 (Fig. 3B, panel c) indicating that both serines are also glycosylated in Sf9 cells (i.e. Ser-29 and -30 of K18, Fig. 1). Verification that Ser-29,30,48 of K18 are the major glycosylation sites in Sf9 cells was obtained by analyzing the glycosylation of the construct Ser-7,8,13 which showed minimal glycosylation (Fig. 3A, lane g) and lack of labeling of peptides 1-3 (Fig. 3B, panel d). Of note, the construct containing Ser-9 mutation had a tryptic glycopeptide pattern identical with wild type K18 (not shown).


Figure 3: Analysis of K18 constructs containing single or multiple mutations at potential glycosylation sites. The numbers represent mutated serines shown as consecutive numbers in Fig. 1. A, K8/18 were immunoprecipitated from Sf9 cells infected with recombinant wild type K8/18 or WT K8/mutant K18. Immunoprecipitates were labeled and then analyzed by SDS-PAGE as described under ``Materials and Methods.'' B, individual labeled K18 bands were isolated and analyzed by tryptic peptide mapping. x = origin where samples were spotted on the cellulose plates. Circled spots represent minor labeled peptides that are inconsistently seen.



Although the major tryptic glycopeptides of K18 expressed in insect cells or as found in wild type human colonic tissue cultured cells are shared, it is possible that variability may occur in the glycosylation sites between the insect and human systems if more than one serine is present within a given glycopeptide. We addressed this possibility using manual Edman degradation of [^3H]galactose-labeled individual glycopeptides of K18 isolated from human or insect cells. As shown in Fig. 1, the tryptic peptides containing Ser-29,30 and Ser-48 have five and two serines, respectively, as potential glycosylation sites in human HT29 cells. Manual Edman degradation of peptides 1 and 2, individually isolated from HT29 cells, showed that most of the released [^3H]Gal-GlcNAc-amino acid label was in cycles 3 and 4 which corresponds to Ser-29 and Ser-30 (Fig. 4, A and B). Similar processing of peptide 3, also isolated from HT29 cells, showed that most of the released label was in cycle 4 which corresponds to Ser-48 of K18 (Fig. 4C).


Figure 4: Identification of the glycosylated residues of human K18 tryptic glycopeptides using manual Edman degradation. K8/18 immunoprecipitates from HT29 cells were labeled with UDP-[^3H]galactose followed by isolation of K18. After tryptic peptide mapping, individual labeled peptides were eluted from the cellulose plates and subjected to manual Edman degradation as described under ``Materials and Methods.'' The counts released from each reaction cycle are represented by the black bars. Peptide numbers correspond to the major glycopeptides shown in Fig. 2. The serines marked by arrows represent the major glycosylation sites on individual peptides based on the counts/min/cycle obtained.



Manual Edman degradation of the identical radiolabeled peptides isolated from Sf9 cells gave a pattern very similar to that in Fig. 4except that cycle 4 of peptides 1 and 2 was 1.6 times higher in counts than cycle 3 (e.g. 728 versus 467 cpm for peptide 1 of K18 from Sf9 cells, not shown), indicating that the specific activity of Ser-30 glycosylation is more than Ser-29 in Sf9 cells, while the opposite is true in HT29 cells. This supports the finding shown in Fig. 3A, where panel c (i.e. mutation at Ser-30 of K18) demonstrates slightly lower incorporation of [^3H]galactose than panel b (i.e. mutation at Ser-29 of K18).

Mutation of K18 Glycosylation Sites Abolishes Glycosylation of the Protein Expressed in Mammalian Cells but Does Not Interfere with Filament Assembly

We compared the glycosylation of WT K8/18 versus WT K8/glycosylation mutant K18 after expression in NIH-3T3 cells, in order to further confirm the K18 glycosylation sites identified using the mutational Sf9 cell expression and biochemical approaches. Transient expression of a K18 Ser-29,30,48 Ala mutant or WT K18, with their obligate heteropolymer WT K8, in NIH-3T3 cells showed that the mutant construct was not glycosylated (Fig. 5, compare lanes 3 and 4) although protein levels of the mutant and wild type K18 were equal (Fig. 5, lanes 1 and 2).


Figure 5: ^3H-galactosylation of K8/18 in NIH-3T3 cells expressing WT K8/18 or WT K8 with the K18 glycosylation mutant. NIH-3T3 cells were transiently transfected with wild type K8/18 (lanes 1 and 3) or wild type K8 and a K18 Ser-29,30,48 Ala mutant (gly-) (lanes 2 and 4). After 3 days, K8/18 were immunoprecipitated, galactosylated with UDP-[^3H]galactose, and then analyzed by SDS-PAGE.



We also asked if obliteration of the glycosylation of K18 interferes with the ability of K8/18 to form filaments after transfection into mammalian cells. As shown in Fig. 6, transfection of WT K18 or a panel of the glycosylation K18 mutants with WT K8 into NIH-3T3 or BHK cells resulted in similar-appearing filaments.


Figure 6: Immunofluorescence of mammalian cells transfected with WT K8 and WT K18 or K18 glycosylation mutants. NIH-3T3 or BHK cells grown on coverslips were transiently co-transfected with WT K8/18 or WT K8 and the indicated K18 single or multiple serine glycosylation mutants. After 3 days, cells were fixed and analyzed by immunofluorescence as described under ``Materials and Methods.'' The numbers represent the mutated serines shown as consecutive numbers in Fig. 1.



DISCUSSION

Identification of the Major Human K18 Glycosylation Sites

This study reports the identification of the major sites of the single O-GlcNAc type of glycosylation for human keratin 18. Several lines of evidence support the assignment of the identified sites. First, a systematic mutational approach coupled with expression in insect cells identified the major tryptic glycopeptides that are shared between K18 isolated from human cells or expressed in insect cells and indicated that Ser-29,30,48 of K18 are glycosylated when K18 is expressed in insect cells. Second, manual Edman degradation of the K18 ^3H-labeled glycopeptides isolated from human or insect cells confirmed the glycosylation positions that were identified using the mutational approach. Third, expression of the glycosylation-negative K18 mutant and lack of its glycosylation in NIH-3T3 cells provided additional evidence for the monosaccharide attachment sites.

A somewhat unexpected observation in this study is that mutation of Ser-29,30 which reside in the same tryptic peptide abolished two radiolabeled spots. Although mutation within one peptide may indirectly affect glycosylation at a distant site, Edman degradation release of radioactivity during the third and fourth cycles for both spots suggests that the two spots correspond to the same peptide. Additional support for this is based on the sequence of human K18 (Refs. 34 and 35 and Fig. 1) which indicates that Ser-29 and Ser-30 are the only Ser-Ser amino acids in K18 that are located at the third and fourth position of a predicted tryptic peptide. Furthermore, the third and fourth amino acids of the predicted tryptic peptides of human K18 lack Ser-Thr or Thr-Ser, and the only Thr-Thr is within the first predicted tryptic peptide of K18 containing amino acids 1-5 (Fig. 1). However, as shown by our mutational analysis, threonine glycosylation does not contribute to the three radiolabeled glycopeptide spots (Table I), and a double mutation of the two threonines in the first tryptic peptide (SFTTR) does not alter K18 glycosylation (not shown). Therefore, the most likely interpretation of our results, although not directly proven, is that spots 1 and 2 ( Fig. 2 and Fig. 3) correspond to differently modified forms of the same peptide. The precise modification that gives rise to two different forms of the same glycopeptide remains to be determined, but phosphorylation is a likely candidate. This is based on phosphorylation of the peptide containing Ser-29,30 (not shown and Ref. 7) and the slight difference of migration of spots 1 and 2 by electrophoresis (horizontal direction of the peptide maps shown in Fig. 2and Fig. 3 ) which is consistent with a charge difference.

Previous indirect biochemical evidence we obtained, based on the generation of alpha-aminobutyrate after beta-elimination and then acid hydrolysis of purified K8/18, suggested that threonine is a likely glycosylation site of K8/18 (12) . However, the results obtained here unambiguously identified three serine but no threonine K18 major glycosylation sites. Our results here do not exclude the possibility that other glycosylation sites of K18 (serine or threonine) that are not accessible to labeling in the presence of UDP-[^3H]galactose and galactosyltransferase may be present.

How Conserved Are the Human K18 Glycosylation Sites?

Although a consensus sequence does not exist for the O-GlcNAc type of cytoplasmic and nuclear protein glycosylation, the K18 glycosylation sites (PVSSAASVY, and ISVSRSTS) show some similarity to already identified sites in other proteins (Table II). For example, most, but not all, identified sites have a nearby proline and/or valine. The glycosylated Ser-48 of K18 has an adjacent valine but no nearby proline. The only other identified O-GlcNAc site that does not have an adjacent proline is ANQLTNDY in talin (36) (Table II). It is not clear yet if one or more GlcNAc transferases is involved in this type of glycosylation and if distinct transferases to serine and threonine residues are present (37) . To date, only one GlcNAc transferase has been characterized (38) . Another common feature of the identified O-GlcNAc sites is a high density of Ser/Thr residues in proximity to the modified residue. These features of the O-GlcNAc type of glycosylation are similar to the general features of the O-linked mucin type of glycosylation (37, 39, 40) .


Table II


The sequence ISVSR is conserved in mouse and human K18 and the sequence PVSSAASV in human K18 is nearly identical with the corresponding sequence PASSAASV in mouse K18 (34, 35) . This suggests that mouse K18 glycosylation is likely to be identical with its human counterpart. The only identified glycosylation sites of intermediate filament proteins are those shown in Table II. However, glycosylation is likely to be a modification that is found in a broad range of IF proteins. For example, the O-GlcNAc type of glycosylation has been characterized in K13 (10) , and glucosamine was shown to be incorporated into a number of epidermal keratins (8, 9) . In addition, there is some evidence for a porcine lamin A-like protein that binds to concanavalin A (41) , although direct evidence for lamin A glycosylation and its nature are not known. Interestingly, the sequence Pro-X^1-X^2-X^3 (where one X = a hydrophobic residue, and two Xcorrespond to Ser/Thr residues or Ser/Thr and any other amino acid) is found in the head domain of a number of intermediate filament proteins. This includes PLSS in human desmin (42) , PLSP in human lamins (43) , and PAST in keratin 16 (44) . Since the sequence Pro-X^1-X^2-X^3 is a glycosylation motif in neurofilament L and M and in K18 (Table II), it is possible that similar motifs in other intermediate filament proteins are also glycosylated.

Potential Functions to Consider for K18 Glycosylation

The few available functional data regarding K8/18 glycosylation are in general exclusionary rather than supportive of several potential tested functional roles. For example, glycosylation does not appear to play a significant role in keratin solubility since the soluble and insoluble K8/18 pools had similar specific activities of glycosylation and similar tryptic glycopeptide maps (45) . In addition, although G(2)/M arrest of HT29 cells using anti-microtubule agents or okadaic acid resulted in a marked increase in K8/18 glycosylation, analysis of G(0)/G(1), S phase, and G(2)/M phase synchronized cells did not reveal any changes in K8/18 glycosylation (46) .

The function of single O-GlcNAcs remains speculative although several lines of evidence indicate that it is likely to be important (reviewed in Refs. 27 and 28). (i) It occurs on molecules that clearly have an important biologic function (Table II), and to that end it may regulate the function of these proteins. For example, wheat germ agglutinin inhibits the transcriptional but not DNA binding capacity of Sp1 presumably by binding the O-GlcNAcs of Sp1 (47). (ii) It is a dynamic modification that has a higher turnover rate than the turnover of the K8/18 proteins (11) , and its levels can change upon cell activation as shown for mouse T cells treated with concanavalin A (29) . (iii) It is a simple modification that may easily come on/off similar to phosphorylation. In fact, most if not all proteins with this modification are also phosphorylated. Examples include K8/18 (12) , eukaryotic peptide chain initiation factor 2-associated p67 polypeptide (48) , RNA polymerase II (49) , clathrin assembly protein AP-3 (50) , and neurofilaments (25, 51) . Interestingly, in the cases that have been studied, it appears that for the most part molecules that are phosphorylated are not glycosylated and vice versa (12, 48, 49; but see Ref. 50 for exception). This indicates that one modification may block the other and/or each modification plays different regulatory functions. For K8/18, it appears that glycosylation and phosphorylation are not related in several systems tested. For example, inhibition of basal phosphorylation of K8/18 in human HT29 cells using staurosporine (12) , or increased phosphorylation of K8/18 during the S or G(2)/M phases of the cell cycle (46) are not associated with altered K8/18 glycosylation. Furthermore, mutation of the major phosphorylation site of human K18 (Ser-52) does not affect K18 glycosylation (7) .

With regard to K8/18 glycosylation, several potential functions may be considered. These include a role in protein-protein interaction at the keratin-keratin level and/or at an associated protein-keratin level. Such roles are attractive possibilities for the O-GlcNAc modification in general since most identified proteins with this modification form multimeric polypeptide complexes (27, 28) . The transfection experiments using wild type or K18 glycosylation mutants did not show an obvious role for K18 glycosylation in filament assembly (Fig. 6). We cannot exclude the possibility that K18 glycosylation plays a role in filament organization not measured by our immunofluorescence assembly assay. The alternative, that of an associated protein-keratin interaction role, is attractive based on the location of the identified modifications in NF-L, M (25) , and K18 (this report). Hence, glycosylation of K18 and NF-L appear to be restricted to the head domain, and glycosylation of NF-M appears to be restricted to the head and tail domains. These are divergent but shared IF protein domains where most of the structural heterogeneity and presumed tissue specific functions reside (15, 16, 17) . Availability of the K18 glycosylation mutants should allow us to begin testing this possibility.

Two additional functions of K8/18 glycosylation that may be considered are subcellular localization and degradation protection roles. For example, prolonged heat stress (52) or mitotic arrest (12) , which ultimately result in cell death, were associated with increased K8/18 glycosylation and phosphorylation at a stage prior to eminent cell death. Although it is not possible to separate glycosylation from phosphorylation effects in these two systems, glycosylation could protect from protein degradation (reviewed in Ref. 53) during impending cell death and basal states. Finally, glycosylated keratin molecules, which appear to be distinct from the phosphorylated species, may be localized in different subcellular compartments. For example, site-specific antiphospho glial fibrillary acidic protein showed that specific sites were phosphorylated within the spatial region proximal to the cleavage furrow during cytokinesis (54) . Although the function(s) of IF protein glycosylation and the single O-GlcNAc type of modification are unclear, the abundance of K8/18 coupled with identification of their glycosylation sites should make addressing functional aspects of this modification more amenable to study.


FOOTNOTES

*
This work was supported by a Veterans Administration Merit Award, National Institutes of Health Grants AA0947 and DK47918 and Digestive Disease Center Grant DK38707. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of an American Heart Association California Affiliate postdoctoral fellowship. To whom reprint requests should be addressed: Palo Alto VA Medical Center, 3801 Miranda Ave., 111-GI, Palo Alto, CA 94304.

To whom correspondence should be addressed: Palo Alto VA Medical Center, 3801 Miranda Ave., 111-GI, Palo Alto, CA 94304.

(^1)
The abbreviations used are: IF, intermediate filaments; GlcNAc, N-acetylglucosamine; K, keratin; PAGE, polyacrylamide gel electrophoresis; WT, wild type.


ACKNOWLEDGEMENTS

We are very grateful to Kris Morrow and Sally Morefield for preparing the figures, to Dr. Dennis Dong for advice regarding manual Edman degradation of ^3H-peptides, and to Linda P. Jacob and Theresa L. Hooper for preparing the manuscript.


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