The type II AFP from rainbow smelt Osmerus mordax, which is a member of the C-type lectin superfamily, was characterized in terms of its Ca2+-binding quaternary structure and the role of
Trang 1Structural and functional characterization of a C-type lectin-like
John C Achenbach1,* and K Vanya Ewart2
1
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada;2NRC Institute for Marine Biosciences, Halifax, Nova Scotia, Canada
Antifreeze proteins (AFPs) are produced by several
cold-water fish species They depress physiological freezing
temperatures by inhibiting growth of ice crystals and, in so
doing, permit the survival of these fish in seawater cooler
than their normal freezing temperatures The type II AFP
from rainbow smelt (Osmerus mordax), which is a member
of the C-type lectin superfamily, was characterized in terms
of its Ca2+-binding quaternary structure and the role of its
single N-linked oligosaccharide The protein core of the
smelt AFP, shown through sequence homology to be a
C-type lectin carbohydrate-recognition domain, was found
to be protease resistant Smelt AFP was also shown to bind
Ca2+, as determined by ruthenium red staining and a
conformational change on Ca2+ binding detected by
intrinsic fluorescence The N-linked oligosaccharide was found to have no effect on protease resistance, dimerization,
or antifreeze activity Thus its role, if any, in the antifreeze function of this protein remains unknown Smelt AFP was also shown to be a true intermolecular dimer composed of two separate subunits This dimerization did not require the presence of N-linked oligosaccharide or bound Ca2+ Smelt AFP dimerization has implications for the effective solution concentration and measurement of its activity This finding may also lead to new interpretation of the mechanism of ice-growth inhibition by this AFP
Keywords: antifreeze protein; C-type lectin; dimerization; glycosylation; rainbow smelt
Antifreeze proteins (AFPs) are produced by many species as
an efficient means of protection from freezing They bind
directly to growing ice crystals and thus inhibit crystal
growth [1] These proteins generate a far greater freezing
point depression than would be predicted from their
colligative properties This is presumed to be due to their
direct interaction with ice crystals The AFPs only have a
colligative effect on the melting point of a solution and
therefore cause a thermal hysteresis This phenomenon is
the basis for quantitative measurement of AFP activity
The AFPs are structurally and evolutionarily diverse but
are all functionally similar in that they bind ice and depress
the freezing point They are found in a variety of insect and
plant species as well as bacteria and fungi, but fish AFPs
were the first and most extensively characterized [2] Among
fish species, five structurally defined types of AFPs have
been identified The antifreeze glycoproteins found in cods
and Antarctic nototheniids are composed of multiple
Ala-Ala-Thr repeats, with a disaccharide linked to each Thr
residue [1] The type I AFPs found in certain flounders and
sculpins are alanine-rich, amphiphilic a helices with a
repeating pattern of Thr residues [3,4] The type III AFPs
have a unique globular fold with one flattened surface thought to take part in ice binding, and are found in northern and Antarctic eel pouts [5] The recently charac-terized type IV AFP is composed of an antiparallel helix bundle homologous to an apolipoprotein, which has been only found in longhorn sculpin, Myoxocephalus octodec-imspinosis [6] Type II AFPs have been shown to be homologous to C-type lectins They are found in the serum
of three teleost fishes: sea raven (Hermitripterus americanus), Atlantic herring (Clupea harengus harengus), and rainbow smelt (Osmerus mordax) [2] They consist of a long form of the C-type carbohydrate-recognition domain and are C-type lectin-like domains (CTLDs) [7–9] This domain consists of a tightly folded hydrophobic core stabilized by three or more disulfide bonds [10]
The type II AFPs of herring and smelt appear to be very closely related but the sea raven AFP is more distinct The smelt and herring type II AFPs exhibit near-equivalent molar antifreeze activity (thermal hysteresis), whereas the sea raven AFP appears far more active in generating thermal hysteresis [11] Moreover, the smelt and herring AFPs are structurally very similar These AFPs share 86% sequence identity in a 126-residue sequence overlap and they have very similar putative signal sequences They share approximately twice the percentage sequence identity with one another than either share with the sea raven protein [7] The smelt and herring AFPs also share the Gln-Pro-Asp (galactose-binding) motif of galactose-binding C-type lec-tins This is the centre of the carbohydrate-binding site in C-type lectins and was shown to be the ice-binding site in herring AFP [12] Like these lectins, which require Ca2+to
be bound before they can bind carbohydrate, the smelt and herring AFPs require Ca2+in order to bind to ice [7,13] Smelt AFP does differ from herring AFP and other fish
Correspondence toK V Ewart, NRC Institute for Marine Biosciences,
1411 Oxford St, Halifax, NS, B3H 3Z1, Canada.
Fax: + 1 902 426 9413, Tel.: + 1 902 426 7620,
E-mail: vanya.ewart@nrc.ca
Abbreviations: AFP, antifreeze protein; BS 3 , bis(sulfosuccinimdyl)
suberate; CTLD, C-type lectin-like domain; Glu-C, endoproteinase
Glu-C.
*Present address: Department of Biochemistry, McMaster University,
Hamilton, Ontario, Canada.
(Received 26 October 2001, accepted 2 January 2002)
Trang 2AFPs, however, in several important respects It has an
18-residue N-terminal extension (GDTGKEAVMTGSSG
KNLT) and an 11-residue C-terminal extension (VNP
EVTPPSIM) not present in herring AFP It also has an
N-linked glycosylation site in the N-terminal extension
sequence [13] Therefore, the goal of this study was to
investigate the structural and functional characteristics of
smelt type II AFP The effects and inter-relationship of
metal ion binding, N-linked glycosylation, and quaternary
structure in this protein were studied in order to better
define its structure and function as an AFP as well as its
relationship to other CTLDs
M A T E R I A L S A N D M E T H O D S
Materials
N-Glycosidase F and endoproteinase Glu-C were obtained
from Roche Molecular Biochemicals (Laval, Canada)
Bis(sulfosuccinimdyl) suberate (BS3) and Gelcode Blue
(Coomassie) stain were obtained from Pierce Chemicals
(Rockford, IL, USA) Sequencing-grade endoproteinase
Glu-C and trypsin were obtained from Promega (Madison,
WI, USA) Ruthenium red was purchased from Fluka
Chemicals (Ronkonkoma, NY, USA) Nitrocellulose
mem-brane (0.45 lm) was purchased from Bio-Rad (Hercules,
CA, USA) All other chemicals were reagent grade
Purification of smelt AFP
Blood plasma was obtained from a population of rainbow
smelt (O mordax) caught in seawater along the
north-eastern coast of Newfoundland on 20 February 1997 and
stored frozen until use Approximately 2.5 mL plasma was
fractionated on a 1· 90 cm S-200 Sephacryl (Pharmacia,
Uppsala Sweden) gel-filtration column Fractions
contain-ing AFP, as determined by SDS/PAGE, were then applied
to a 1· 30 cm phenyl–Sepharose hydrophobic interaction
column (Amersham–Pharmacia), and eluted in 20 mMTris/
HCl, pH 8.0, buffer with a continuous descending (1–0M)
NaCl gradient Fractions containing AFP, as identified by
SDS/PAGE, were pooled and lyophilized The lyophilized
sample was reconstituted in a minimal volume of 0.1M
NH4HCO3buffer, pH 8.0, desalted using a PD-10 column
(Amersham Pharmacia), lyophilized, and stored at)20 °C
Preparation of deglycosylated smelt AFP
Lyophilized N-glycosidase F was reconstituted to 1 UÆlL)1
in water Lyophilized smelt AFP was dissolved in 25 mM
Hepes, pH 7.8, to a final concentration of 4 mgÆmL)1 Then
10 U N-glycosidase F was added to 100 lL of smelt AFP
solution and incubated at 37°C; a further 5 U
N-glyco-sidase F added 4 h later, and again after an overnight
incubation A further 8 U was added over a period of 4 h
followed by a second overnight incubation at 37°C The
reaction was considered complete after SDS/PAGE analysis
of the reaction mixture revealed no detectable glycosylated
smelt AFP using Gelcode Blue staining A parallel control
reaction was carried out by adding equivalent volumes of
water to 100 lL of the smelt AFP solution, and incubating
at 37°C for the same amount of time
Ruthenium red staining Ruthenium red dye binding was evaluated following the method of Charuk et al [14] with minor modifications Deglycosylated smelt AFP and untreated smelt AFP samples (both 4.5 lg per lane) were run on an SDS/15% polyacrylamide gel under nonreducing conditions and then electrophoretically transferred to a 0.45-lm nitrocellulose membrane A blot section was incubated at 4°C for
15 min in staining buffer (20 mM Hepes, pH 7.8,
10 mgÆmL)1 ruthenium red), followed by a 15-min wash
at 4°C in wash buffer (20 mM Hepes, pH 7.8) An identical section was treated the same way except
100 mMCaCl2was present in both the staining and wash buffer Amido black staining was used to confirm protein presence and to detect bands not stained by the ruthenium red dye
Analysis of antifreeze activity Antifreeze activity was quantitated as thermal hysteresis, which is the difference between the melting and freezing point of a solution Thermal hysteresis was measured by monitoring ice crystal behavior using a nanoliter osmometer (Clifton Technical Physics, Hartford, NY, USA) Five measurements were taken on 130-lMsolutions of deglycos-ylated and untreated smelt AFP in 25 mMHepes, pH 7.8, using water as a blank As a control, the thermal hysteresis
of a solution of N-glycosidase F was measured in 25 mM Hepes, pH 7.8 Results were expressed as mean ± SE Photographs of ice crystals viewed during these measure-ments were taken at a magnification of 200·
Protease protection assays on smelt AFP Protease protection assays to compare deglycosylated and untreated smelt AFP were performed as previously described [15] with minor modifications Reaction volumes
of 16 lL containing 4.8 lg of either untreated or deglycos-ylated smelt AFP in 10 mMHepes, pH 7.8, and either 2 mM EDTA or 20 mMCaCl2 were incubated for 30 min This was followed by the addition of protease (0.1 mgÆmL)1final concentration), or an equal volume of water, after which all samples were incubated for 3 h at room temperature An aliquot of each reaction mixture was resolved by SDS/ PAGE (15% gel) under reducing conditions, and stained with Gelcode Blue
In vitro chemical cross-linking Untreated and deglycosylated smelt AFP samples in HCS buffer (25 mM Hepes, pH 7.8, 150 mM NaCl, 10 mM CaCl2) or HES buffer (10 mM EDTA in place of CaCl2)
at a final AFP concentration of 33 lM, were aliquoted into the wells of a microtiter plate and left undisturbed for
15 min at room temperature A serial dilution of BS3 dissolved in either HCS or HES buffer was performed, and aliquots of each dilution were added to corresponding AFP-containing wells The reaction mixtures were incubated at room temperature for 1 h, then resolved by SDS/PAGE (15% gels) under reducing conditions and stained with Gelcode Blue
Trang 3Determination of molecular mass by gel-filtration
chromatography
A TosoHaas 4-lm particle size TSK SuperSW 2000 column
(4.6· 300 mm) was equilibrated in HCS buffer and run at a
flow rate of 0.3 mLÆmin)1using an HPLC (Waters) The
column was calibrated using protein molecular-mass
stand-ards Approximately 15 lg untreated smelt AFP was
applied to the column in duplicate runs Similar samples
of deglycosylated AFP were applied as well All proteins
were detected by monitoring A230
Fluorescence measurements
Intrinsic fluorescence was measured using an Aminco
Bowman series 2 spectrofluorimeter at room temperature
Untreated and deglycosylated smelt AFP samples were
diluted to equimolar concentrations in 25 mM Hepes,
pH 7.8, containing 1 mM EDTA Emission spectra were
recorded with a 2-nm/s scan rate in duplicate for each trial
with an excitation wavelength of 280 nm (4 nm bandpass)
CaCl2was added by pipetting a 0.5-Msolution directly into
the sample cuvette with thorough mixing followed by a
10 min incubation at room temperature A 0.5-Msolution of
EDTA (pH 8.0) was added in a similar fashion to test the
reversibility of any Ca2+effect Spectra were corrected for
the resultant dilutions
R E S U L T S
Deglycosylation of smelt AFP
To study the effect of the N-linked oligosaccharide of smelt
AFP, it was removed from the protein enzymatically using
N-glycosidase F The reaction evaluated using SDS/PAGE
was found to result in a band of reduced size (17 kDa)
compared with the untreated band (22 kDa) (results not
shown) The 17-kDa molecular mass corresponds to the
17.4-kDa calculated mass of the herring AFP sequence
(from cDNA) with the predicted signal sequence removed
This result implies that the smelt AFP has no prosequence
and consists of the complete sequence predicted from
cDNA minus the signal
Ruthenium red staining
To detect Ca2+binding by smelt AFP, both untreated and
deglycosylated smelt AFP were blotted as discussed in
Materials and methods and stained with a 10 mgÆmL)1
solution of ruthenium red This dye was shown to bind both
the untreated and deglycosylated smelt AFP (Fig 1) as well
as the known Ca2+-binding molecular mass standard
b-lactoglobulin (not shown) The specificity of binding is
demonstrated by the absence of detectable ruthenium red
staining when CaCl2is added to the staining and washing
buffer (Fig 1)
Measurement of antifreeze activity
Antifreeze activity was measured on equimolar amounts of
deglycosylated and untreated smelt AFP to determine
whether the N-linked oligosaccharide had any effect on this
activity A faceted ice-crystal morphology signifies the
presence of antifreeze activity The rounded ice crystal formed in the control sample containing only N-glycosi-dase F shows that the enzyme does not display antifreeze activity (Fig 2) Equimolar solutions of deglycosylated and untreated smelt AFP displayed similar faceted ice crystal morphology (Fig 2) The antifreeze activity of the
untreat-ed and deglycosylatuntreat-ed samples, quantitatuntreat-ed as thermal hysteresis, were 0.027 ± 0.003 and 0.026 ± 0.003, respect-ively These are not significantly different (P < 0.001) As expected, the enzyme control sample showed no activity, with a hysteresis value of)0.001 ± 0.002
Protease protection assays
To determine whether Ca2+or the N-linked oligosaccha-ride has any effect on the protease susceptibility of smelt AFP, both untreated and deglycosylated smelt AFP were digested with Glu-C and trypsin in the presence and absence
of Ca2+ Both enzymes generated some proteolysis near the N-terminus and/or C-terminus of the protein leaving a large central AFP fragment intact The sizes of the digestion fragments were all greater than 14 kDa, which is the approximate size of the core CTLD The addition of Ca2+
at a concentration of 20 mMhad no effect on the proteolysis seen with either deglycosylated or untreated smelt AFP (Fig 3A,B) This suggests that the CTLD of smelt AFP is protease resistant both in the presence and absence of Ca2+ and with and without the N-linked oligosaccharide Similar digestion patterns were also seen in both the untreated and deglycosylated smelt AFP trials using either protease Digestion of either deglycosylated or untreated smelt AFP formed products that were 1 kDa and 2 kDa smaller than undigested smelt AFP The size differences between the digestion fragments of untreated and deglycosylated AFP
Fig 1 Ruthenium red staining of smelt AFP Ruthenium red (RR) staining of deglycosylated (D) smelt AFP and untreated (U) smelt AFP was carried out as described in Materials and methods Blots shown are stained with amido black (Amido), ruthenium red (RR)
or ruthenium red in the presence of 100 m M CaCl 2 (RR + Ca 2+ ) Molecular-mass marker sizes (kDa) are indicated.
Trang 4are all 5 kDa, which corresponds to the apparent size of the
carbohydrate determined in a separate experiment (Fig 1)
Taken together, these results indicate that the proteases
digest both untreated and deglycosylated forms of smelt
AFP in close, if not identical, locations These results also
indicate that protease digestion under these conditions does
not cleave between the Asn residue carrying the N-linked
oligosaccharide and the core CTLD Bands corresponding
to Glu-C and trypsin enzymes were identified on the basis of
molecular mass The bands smaller than 14 kDa in the
trypsin digests were shown to be trypsin autolysis products
in a separate control experiment containing trypsin alone
(not shown)
Intrinsic fluorescence
Modulation of protein conformation on addition of Ca2+
was monitored by intrinsic fluorescence Excitation at
280 nm produced an emission spectrum with a kmax of
343 nm from 30 lMsamples of both untreated (Fig 4A)
Fig 2 Analysis of antifreeze activity Antifreeze activity was evaluated
qualitatively by monitoring ice crystal morphology in solutions
con-taining deglycosylated and untreated smelt AFP (both 130 l M ) as
described in Materials and methods The top row of photos is of ice
crystals with their c axis in the plane of the page The bottom row
shows ice crystals with their c axis normal to the page.
Fig 3 Proteolytic digestions of deglycosylated and untreated smelt AFP in presence and absence of Ca2+ Both deglycosylated and untreated smelt AFP were incubated with protease in the presence or absence of 20 m M Ca2+as described in Materials and methods All reaction products were analyzed by SDS/PAGE and stained with Gelcode Blue (A) AFP digested with Glu-C Bands corresponding to Glu-C, untreated (UT) AFP (UT AFP), and deglycosylated AFP (DG AFP) are identified Proteolytic fragments are seen as lower-molecular-mass bands in Glu-C-containing lanes (B) AFP digested with trypsin Bands corresponding to trypsin, untreated AFP (UT AFP), and deglycosylated AFP (DG) are identified Bands smaller than 14 kDa in lanes containing trypsin were shown to be trypsin autolysis products (trypsin-only digest not shown) Molecular-mass marker sizes (kDa) are indicated.
Fig 4 Effect of Ca2+on the intrinsic fluorescence of deglycosylated and untreated smelt AFP (A) Emission spectra of intact (untreated) AFP (UT AFP) (B) Emission spectra of deglycosylated AFP (DG AFP) Spectra were recorded for solutions of 30 l M smelt AFP in 25 m M
Hepes, pH 7.8, containing 1 m M EDTA, before and after the addition
of CaCl 2 (10 m M ) Each spectrum shown is the average of two suc-cessive recordings All measurements were taken with excitation at
280 nm and a 2-nmÆs)1scan rate at ambient temperature.
Trang 5and deglycosylated (Fig 4B) smelt AFP The emission
intensity was shown to increase significantly in both samples
on addition of Ca2+to a final concentration of 10 mM The
effect was fully reversible in both samples, with the addition
of EDTA to a final concentration of 18 mM(not shown)
No blue or red shift was observed in the emission spectra of
either sample on addition of Ca2+or EDTA
Detection of dimerization
To determine the quaternary structure of smelt AFP in
solution, AFP (33 lM) was incubated in the presence of a
range of concentrations of BS3, a water-soluble
homo-bifunctional cross-linking agent, in the presence of 10 mM
CaCl2 In the absence of BS3, a 22-kDa band corresponding
to the smelt AFP monomer was observed (Fig 5A) A BS3
concentration of 0.16 mM was sufficient to generate the
accumulation of a larger protein form With increasing
concentrations of BS3, the disappearance of the monomer
band corresponded to the gradual increase in intensity of a
higher-molecular-mass band The apparent molecular mass
of this band was 38 kDa, which is substantially higher than
the molecular mass of the monomer band, indicating the
presence of a dimer The expected mass of a smelt AFP
dimer is 44 kDa but, on SDS/PAGE, the covalently
cross-linked dimer would be expected to migrate faster than its
actual mass would predict because of limited linearization in
SDS The value of 38 kDa is consistent with such a dimer
band A small amount of a high-molecular-mass aggregate
was evident at the top of the SDS/PAGE gel lane containing
the sample with the highest concentration (20 mM) of BS3
This appears to be an artefact of high cross-linker
concen-tration and was not taken as evidence of any larger protein
aggregate To determine whether the oligosaccharide plays a
role in dimerization, the experiment was repeated on
deglycosylated smelt AFP (Fig 5B) Dimerization was
evident with monomer bands of 18 kDa and dimer bands
of 33 kDa To examine whether Ca2+binding was required
for dimerization, smelt AFP was incubated with BS3 in
buffer containing 10 mMEDTA instead of CaCl2 In the
absence of added Ca2+, dimerization was again observed (Fig 5C)
The molecular mass of smelt AFP determined by HPLC gel-filtration analysis was 50 kDa (Fig 6) As the molecular mass of the protein on SDS/PAGE is 22 kDa, the value obtained by gel filtration is consistent with a dimer There was no evidence of larger aggregates, as determined by absorbance at 230 nm, nor were there any detectable peaks corresponding to the monomer size (22 kDa) These results are in agreement with the cross-linking experiments indica-ting that the native smelt AFP is fully dimerized Deglycos-ylated AFP was also analyzed on the same column and found to have a molecular mass of 41 kDa, which also indicates dimer formation (not shown)
Fig 5 Chemical cross-linking of smelt AFP Smelt AFP (33 l M ) was cross-linked using BS3as described in Materials and methods After the 1 h incubation, reaction contents were analyzed using SDS/PAGE under reducing conditions and stained using Gelcode Blue Lanes 1–9 in all gels correspond to BS 3 concentrations of 0, 0.16, 0.3, 0.6, 1.25, 2.5, 5, 10, and 20 m M , respectively (A) Intact AFP in the presence of CaCl 2 (B) Deglycosylated AFP in the presence of CaCl 2 (C) Intact AFP in the presence of EDTA Positions of dimers (D) and monomers (M) and the molecular-mass marker sizes (kDa) are indicated.
Fig 6 HPLC gel-filtration analysis of smelt AFP Smelt AFP was applied to the column under the conditions described in Materials and methods The elution times of molecular-mass marker proteins (kDa) are indicated by arrows.
Trang 6D I S C U S S I O N
Intermolecular dimer formation by the smelt AFP makes it
unique among the fish AFPs Other AFP types, such as type
I, IV, and the antifreeze glycoproteins, appear to exist as
monomers Similarly, type III AFP is monomeric, and
shown not to self-associate when binding ice [16] A type III
AFP from an Antarctic eel pout (Rhigophila dearborni)
contains a tandem repeat of the domain that comprises
other type III AFPs on a single chain [17] However, the
type III AFP, referred to as an intramolecular dimer, is in
effect a monomer (single polypeptide chain) and not a true
intermolecular dimer The AFPs of winter rye (Secale
cereale) were found to form larger intermolecular
com-plexes, although the precise subunit composition in the
hetero-oligomers containing different AFPs from this plant
is unknown [18] The dimerization of smelt AFP is
intriguing because the homologous sea raven type II AFP
was found to exist as a monomer in solution [19]
Dimerization of smelt AFP is unlikely to increase activity
by increased ice binding because only a dimer with perfectly
aligned ice-binding sites would have enhanced binding to ice
and this is not likely to be the case This may be relevant to
the calculation of smelt AFP activity The activity of smelt
AFP is about one-third of that of sea raven AFP on a
monomer concentration basis [11] As sea raven and herring
AFPs bind to ice at a distinct site on the CTLD [12,21], it is
possible that the activity difference simply reflects the
difference in ice binding However, if the dimerization of
smelt AFP effectively prevents one subunit from binding ice,
this could account for some or all of the difference in activity
calculated on a monomer basis The gel-filtration and
cross-linking results shown here suggest that smelt AFP is fully
dimerized under physiological conditions, with very low or
undetectable levels of monomeric protein Because smelt
AFP is fully dimerized, it would be more appropriate to
calculate its activity on the basis of dimer molarity than on a
monomer basis
Although the dimerization site of smelt AFP cannot be
determined from the results of this study, it is unlikely that
the dimerization is due to intermolecular disulfide bonds
Smelt AFP migrates on SDS/PAGE under nonreducing
conditions with a monomeric size, as shown in the
ruthenium red-binding assay (Fig 3) In addition, all of
the cysteine residues are conserved among the three type II
AFPs and were shown to form intramolecular disulfide
bonds in the monomeric sea raven AFP structure [8]
Because it was possible to chemically cross-link the smelt
AFP dimers using the homobifunctional cross-linking agent
BS3, dimerization must result in the placement of two
primary amines, from either lysine side chains, or the
N-terminus, a maximum distance of 11.4 A˚ apart This
value corresponds to the spacer arm length of the BS3
Several of the soluble C-type lectins associate to form dimers
[22–25] Therefore, the dimerization of smelt AFP is
consistent with other members of the C-type lectin
super-family and is well in keeping with the soluble lectins among
them By analogy with other C-type lectins known to
dimerize, there could be several types of dimerization
surfaces [22,23] Dimerization or multimerization are
com-mon characteristics of C-type lectins, especially those
involved in the acute-phase response of host immune
defense such as the collectins In such cases, multimerization
serves to enhance the avidity and pattern recognition of such lectins towards pathogen carbohydrate structures [26] The inorganic dye ruthenium red has been shown to selectively bind to Ca2+-binding sites of several proteins, including the herring AFP [12–15] Like the herring AFP, smelt AFP was shown to bind the Ca2+ analogue, ruthenium red, suggesting the presence of a Ca2+-binding site Further evidence for direct Ca2+binding by smelt AFP was obtained using intrinsic fluorescence An increase in intrinsic fluorescence emission intensity was shown to result from Ca2+addition, consistent with the result of similar experiments on herring AFP [15] This increase indicates a change in the environment of tryptophan residues in the protein on Ca2+addition, which is normally indicative of a conformational change The number of bound Ca2+ions per smelt AFP monomer remains unknown However, because smelt AFP is identical with that of herring in the positions corresponding to lectin Ca2+-binding sites [7], it is reasonable to suggest that the smelt AFP binds a single
Ca2+in the same way as the herring AFP [15]
In comparison with other C-type lectins and herring AFP, smelt AFP appears to be a remarkably protease-resistant protein in both the presence and absence of Ca2+ ions Digestion with trypsin or endoproteinase Glu-C resulted in the formation of two slightly smaller digestion fragments regardless of whether or not Ca2+was present The large size of the digestion fragments indicates that most
of the smelt AFP is unavailable for protease digestion under the conditions tested The sizes of the digestion fragments were on average only 1–2 kDa smaller than the undi-gested smelt AFP, suggesting that cleavage of only the extended N-terminus and C-terminus occurred, leaving the remaining CTLD These results are consistent with the hypothesis that smelt AFP monomer is composed primarily
of a single CTLD However, in similar studies, the CTLD-containing herring AFP and chicken hepatic lectin did not show the same level of protease resistance in the absence of bound Ca2+ [15,27] The dimerization shown to occur between smelt AFP monomers may enhance protease resistance by shielding residues in the dimerization interface
A conformational change shown to occur in herring AFP
on Ca2+binding generated a shift from protease sensitivity
to protease resistance on Ca2+binding [15] However, in the present investigation of smelt AFP, there was evidence for a protease-resistant CTLD core in the absence Ca2+ The addition of Ca2+ afforded no further protection from proteolysis It is clear from the fluorescence experiments that apo-(smelt AFP) undergoes a conformational change in response to Ca2+binding, which in other lectins and herring AFP is necessary for protease resistance It is highly unlikely that sites susceptible to proteolysis in Ca2+-free herring AFP and related C-type lectins would be less so in smelt AFP as a result of any residue differences because equiv-alent protease resistance and fragment patterns were evident when trypsin was used instead of Glu-C The cross-linking experiments revealed that smelt AFP dimerizes in the presence or absence of EDTA, indicating that the protein remains dimerized in the absence of bound Ca2+ions This may protect susceptible surfaces from protease digestion in smelt AFP, with or without Ca2+
An interesting difference between smelt AFP and the other known AFPs of fish, including the other type II AFPs,
is the presence of an N-linked oligosaccharide located on
Trang 7Asn18 [19] The biological roles of the N-linked
oligosac-charides from many proteins have been studied (reviewed in
[28,29]) N-linked oligosaccharides have been shown to
enhance the thermal stability of proteins, modulate and
stabilize protein secondary structures such as b turns,
mediate intercellular transport of polypeptides, modulate
protein half-life, and facilitate protein–protein interactions
[28–31] To examine possible roles of the N-linked
oligo-saccharide of smelt AFP in native protein structure and
function, deglycosylated and untreated smelt AFP were
compared in terms of dimerization, Ca2+binding, protease
resistance, and antifreeze activity Enzymatic cleavage with
N-glycosidase F resulted in an apparent reduction in
molecular mass of 5 kDa The removal of the
carbohy-drate moiety had no effect on any of the characteristics of
smelt AFP that were investigated However, the possibility
of alternative and untested roles of the N-linked
oligosac-charide such as enhanced protein half-life or recognition by
endogenous lectins in vivo cannot be ruled out
Rainbow smelt are unique among bony fishes in that they
have been shown to produce large amounts of glycerol
(up to 0.4M) in response to subzero temperatures At these
concentrations, glycerol generates a substantial colligative
freezing point depression and depresses the body freezing
temperature of the smelt along with the noncolligative
activity of the smelt AFP [32] Although smelt AFP clearly
contributes to the depression of the serum freezing
tem-perature, it does not appear to be the major contributor to
this effect It would therefore be interesting to determine
whether the AFP has a separate undiscovered activity in
smelt plasma, in which the N-linked oligosaccharide and
dimeric character play more central roles
Smelt AFP has been characterized in terms of Ca2+
binding, function of the N-linked oligosaccharide, and
quaternary structure This study demonstrates that Ca2+
imparts a conformational change when bound to smelt AFP
in the same way as for herring AFP However, unlike
herring AFP, the core CTLD of smelt AFP is protease
resistant even without Ca2+bound Smelt AFP is unusual
among general AFPs in that it is N-glycosylated and
dimeric The smelt type II AFP does not bind common
carbohydrates as many lectins do [7], but its similarity to the
soluble C-type lectins is evident in its structural
character-istics and function The mechanistic implications of the
observed dimerization on smelt AFP ice binding requires
further study
A C K N O W L E D G E M E N T S
We thank Devanand Pinto (NRC IMB) for helpful review of the
manuscript We also thank a number of IMB colleagues for their kind
assistance, including Robert Richards for helpful discussion, Shawna
MacKinnon for use of her HPLC, Denise LeBlanc for loan of a size
exclusion HPLC column, Steve Locke for trypsin, and Neil Ross for
time on his fluorimeter and patient instruction in software use for the
instrument We are grateful to A/F Protein Canada Inc for their
generous donation of smelt blood plasma This research was supported
by NRC IMB This is NRC publication number 42345.
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