(Received for publication, January 13, 1995; and in revised form, March 20, 1995 )
From the
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
H-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.
Keratin intermediate filaments (IF),(
)
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.
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
NHHCO
) 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
HANCE and then
exposed for 7-14 days.
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-[H]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.
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).
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
[H]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
[
H]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-[H]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
[H]galactose than panel b (i.e. mutation at Ser-29 of K18).
Figure 5:
H-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-[
H]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.
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
-aminobutyrate after
-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-[
H]galactose and galactosyltransferase may be
present.
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-X
-X
(where one X
= a hydrophobic
residue, and two X
correspond 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
-X
-X
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.
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/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.