antibodies are multifunctional glycoproteins , 
 able to bind antigens 
 through variable fab domains and cellular receptors via the constant 
 fc region .
this dual functionality enables the recruitment of the 
 cellular immune system to sites of infection by antibody - dependent 
 cellular cytotoxity ( adcc ) and antibody - dependent cellular phagocytosis 
 ( adcp ) , and can lead to the localized activation of the complement 
 system .
glycan and protein engineering of the fc domain can generate 
 therapeutic monoclonal antibodies with tailored receptor binding functionality .
in contrast to chemical and chemoenzymatic methods to modulate glycan 
 structures , we use glycosidase inhibitors and a cell line deficient in a glycosyltransferase 
 to generate antibody glycoforms containing specific carbohydrate structures .
the fc region of immunoglobulin g ( igg ) is a homodimer consisting 
 primarily of heavy chain c2 and c3 domains .
protein 
 interface , occluding over 1100  of protein surface , and adopt rigid conformations that exhibit little 
 structural variation .
in contrast , the 
 c2 domain protomers have only been observed to interact via 
 glycan  glycan contacts between opposing n - linked chains at 
 asn297 .
glycan - mediated maintenance of the spacing between 
 the c2 domains is critical for cellular fc receptor 
 ( fcr ) binding , which occurs asymmetrically at the tip of the 
 c2 domains and lower hinge region .
deglycosylation , for example , by bacterial endoglycosidases , leads 
 to disruption of c2 spacing and significantly impairs fcr 
 binding .
the impact of asn297 glycosylation 
 upon fc structure is not limited 
 to influencing c2 spacing .
these glycan  protein contacts 
 are believed to limit both the processing by golgi - resident glycosyltransferases 
 and the conformational freedom of the glycan .
this model is supported by an nmr study , which proposes that fc 
 glycans exist in an equilibrium with an approximately equal proportion 
 of a  free 
state , with highly mobile glycans , and a 
 less mobile  bound  state , observable by x - ray crystallography , 
 with ordered protein  glycan interactions less accessible to 
 enzymatic processing .
the glycosylation exhibits limited processing and consists of a 
 predominantly biantennary complex - type framework with partial occupancy 
 of galactose , core 16-linked fucose , low levels of 
  bisecting  glcnac , and sialic acid .
this limited processing is in contrast to the highly sialylated 
 complex - type glycosylation typically observed on secreted glycoproteins .
the human fcrs ( fcri , fcriia , 
 fcriib , 
 and fcriiia ) display binding properties dependent upon the 
 presence and composition of the fc glycan .
for example , afucosylated antibody glycoforms , which may find utility 
 in anticancer treatment , are inflammatory and exhibit enhanced adcc 
 due to elevated binding to the activatory fcriiia .
in contrast , anti - inflammatory igg glycoforms 
 display increased levels of terminal sialylation and are under investigation 
 for enhanced intravenous immunoglobulin therapy .
biosynthetic fc precursors have also been investigated 
 for therapeutic 
 applications due to their altered fcr - dependent effector functions .
monoclonal antibodies found in the early steps of carbohydrate maturation 
 including oligomannose- or afucosylated hybrid - type glycans , for example , 
 display increased affinity for fcriiia and enhanced adcc functionality , albeit with potentially elevated serum clearance . here , we have generated and characterized a panel of such glycoform 
 intermediates and present the crystal structure of the key precursor 
 bearing hybrid - type glycosylation . in the context of the biosynthetic 
 pathway of n - linked carbohydrates , this glycoform represents the intermediate 
 formed between the immature oligomannose and the native , complex - type 
 states .
this fc glycoform , generated 
 by recombinant mammalian protein expression in the presence of the 
 golgi -mannosidase ii inhibitor , swainsonine , was crystallized and subjected to x - ray crystallographic analysis 
 to 2.4  resolution .
together 
 with thermostability analyses , the structure provides a model for 
 the conformational transitions that igg fc undergoes upon glycoprotein 
 maturation and provides a template for the structure - guided engineering 
 of therapeutic antibodies .
a 
 panel of igg1 fc glycoforms , corresponding to key stages of the mammalian 
 n - linked biosynthesis after calnexin / calreticulin - mediated protein 
 folding , was generated using either a lectin - resistant 
 cell line deficient in glycosyltransferase activity or by the use 
 of glycosidase inhibitors ( figure 1 ) .
the n - linked 
 glycosylation processing pathway ( left ) and maldi - tof 
 ms analysis of associated igg1 fc glycoforms ( right ) . following protein 
 folding and hydrolysis of the glucose cap , glycoforms were isolated 
 by stalling the pathway at sequential stages of biogenesis . ( a )
( b ) the man5glcnac2 glycoform resulted from expression in a glcnac transferase 
 ( gnt ) i - deficient cell line .
( c ) hybrid - type glycosylation resulted 
 from expression in the presence of the golgi -mannosidase ii 
 ( gmii ) inhibitor , swainsonine .
( d ) complex - type glycosylation resulted 
 from activity of golgi - resident glycosyltransferases .
the following symbols were used to represent glycans and are shown as a key in panel d : yellow  , 
 galactose ; blue  , glcnac ; green  , man ; red  
 with black dot , fucose ; pink  , sialic acid .
linkage positions 
 are shown by the angle of the lines linking the sugar residues ( vertical 
 line , 2-link ; forward slash , 3-link ; horizontal line , 4-link ; back 
 slash , 6-link ) .
we isolated igg1 fc bearing man9glcnac2 , 
 man5glcnac2 , hybrid- , and complex - type glycan 
 structures .
these glycoforms are generally representative of the carbohydrates 
 appearing in the er , and the early , medial , and late golgi apparatus , 
 respectively .
the n - linked glycosylation of each 
 fc glycoform was assessed by positive ion matrix - assisted laser desorption / ionization 
 ( maldi ) time - of - flight ( tof ) mass spectrometry ( ms ) of enzymatically 
 released glycans ( figure 1 ) .
expression of 
 fc in the presence of the er and golgi 12-mannosidase 
 inhibitor , kifunensine , resulted in a largely homogeneous man9glcnac2 glycan ( m / z = 1905.6 ; figure 1a ) with limited processing 
 to the man8glcnac2 derivative ( m / z = 1743.6 ; figure 1a ) . 

expression of the fc region in gnt i - deficient human embryonic kidney 
 ( hek ) 293s cells resulted in a similarly 
 homogeneous spectrum dominated by man5glcnac2 glycans ( m / z = 1257.4 ; figure 1b ) .
the next glycoform in the n - linked biosynthetic 
 pathway , the hybrid - type glycan , was isolated using the golgi -mannosidase 
 ii inhibitor , swainsonine .
swainosine prevents hydrolysis of the mana and manb saccharides of the  6-arm  
 of the trimannosyl core .
however , this inhibition 
 does not impede the addition of a glcnac residue to the  3-arm  
 man4 by gnt i or subsequent structural elaborations .
consistent with this mode of action , maldi - tof 
 ms analysis of the n - linked glycans of igg1 fc expressed with 10 m 
 swainsonine revealed an heterogeneous spectrum of putative hybrid - type 
 glycans indicating variable terminal 14-linked galactose , 
  bisecting 
14-linked glcnac , and a population 
 of sialylated hybrid - type glycans ( m / z = 2081.7 ; figure 1c ) .
finally , fc bearing 
 complex - type glycans were generated using hek 293 t cells with no inhibitors 
 present .
maldi - tof ms analysis revealed biantennary complex - type glycans 
 with variable terminal galactose ( figure 1d ) . 

this spectrum is consistent with previous observations that fc glycosylation 
 is highly protein - directed and substantially 
 less processed than other glycoproteins similarly expressed using 
 the phlsec expression vector in hek 293 t cells .
levels of both galactose and sialic acid in the hybrid - type 
 spectrum 
 were higher than those observed for the complex - type glycoforms .
as 
 sialic acid can alter the ionization efficiency of glycans in mass 
 spectrometry , we also subjected the hybrid 
 and complex - type glycans to electrospray ionization ( esi ) mass spectrometry 
 ( figure 2 ) .
ms of the glycans released from the fc glycoform bearing 
 complex - type glycans ( figure 2b ) .
in contrast , 
 the spectrum of glycans deriving from fc expressed in the presence 
 of swainsonine contained a prominent peak at m / z 2035.7 corresponding to a sialylated core - fucosylated 
 hybrid - type glycan ( figure 2a ) .
negative ion electrospray 
 mass spectra of recombinant igg1 fc n - linked 
 glycans following expression in the ( a ) presence and ( b ) absence of 
 the golgi -mannosidase ii inhibitor , swainsonine .
neutral glycan 
 ions are [ m + h2po4 ] , sialylated 
 glycans are [ m  h ] .
isomeric assignments 
 were determined by esi  ms / ms ( figure 3 ) .
the presence of a minor population of man4-based hybrids 
 was detected in the fragmentation spectra by very low abundance a3 ions at m / z 424 ( figure 3b , d ) .
isomeric assignments of the hybrid - type structures 
 were determined 
 by negative - ion esi ms / ms of the enzymatically released glycans .
the 
 fragmentation spectra of the most abundant species are presented in 
 figure 3 .
the m / z values of the  d - type ions  , as defined by harvey , are a signature of the 6-arm .
these d - type ions 
 are annotated in the spectra and are formed by the formal loss of 
 the 3-arm and the fucosylated di - n - acetylchitobiose 
 core .
similarly , the d-type ions , formed by cleavage of the 
 6-arm , reveal the cluster of mannose residues on the 6-arm .
bisecting 
 glcnac residues are revealed by an abundant [ d  221 ] ion at m / z 629 and the virtual 
 absence of a d - type ion ( figure 3b ) .
the absence of an ion at m / z 306 shows that the sialic acid residue is 23-linked 
 ( figure 3a ) .
negative ion fragmentation 
 spectra of the major n - linked glycans 
 from the hybrid - type gycoform of igg1 fc . neutral glycan ions are 
 [ m + h2po4 ] , while the sialylated 
 glycan forms a [ m  h ] ion .
( a ) sialylated , 
 fucosylated hybrid glycan ( man5gal1glcnac3fuc1neunac1 ) , m / z 2035.7 .
( b ) bisected , fucosylated hybrid glycan ( man5glcnac4fuc1 ) , m / z 1883.6 .
( d ) agalactosylated , fucosylated hybrid 
 glycan ( man5glcnac3fuc1 ) , m / z 1680.5 . the nomenclature describing 
 fragmentation ions follows that of domon and costello and is distinct from the carbohydrate residue labels ( figure 1 ) . to assess the level of sialic acid in the hybrid - type 
 glycans
, 
 we expressed an intact igg1 antibody hybrid - type glycoform and subjected 
 the fluorescently derivatized glycans to normal - phase hplc .
this analysis 
 revealed that 20% of these swainsonine - induced hybrid - type glycans 
 were sialylated ( figure 4a ) .
ms data of igg1 fc glycans , no 
 sialylated structures were observed in igg produced in the absence 
 of swainsonine ( figure 4b ) .
swainsonine 
 is not known to affect galactosyltransferase or sialyltransferase 
 activity , as confirmed by the similar glycan profiles of the same 
 glycoproteins expressed in hek 293 t cells in the presence of swainsonine 
 and in hek 293 t lec36 cells that are devoid of golgi -mannosidase 
 ii activity .
similarly , there was no 
 evidence of increased terminal processing upon disruption of golgi 
 -mannosidase ii activity as compared to glycoproteins expressed 
 in hek 293 t cells .
therefore , the increased 
 abundance of galactose and sialic acid residues in the 3-arm of the 
 hybrid - type glycoform , as compared to the complex glycoform , may be 
 indicative of increased steric accessibility of the fc glycans to 
 processing enzymes .
one explanation 
 for the increased 3-arm processing of the hybrid - type glycoform as 
 compared to that of the complex - type glycan is that the accessibility 
 of the glycans is influenced by composition and structure of the glycan 
because of the highly processed composition of the hybrid - type 
 glycoform , we hypothesized it would exhibit distinct glycan  protein 
 packing interactions with altered stability .
we assessed our panel 
 of fc glycoforms by differential scanning flourimetry ( figure 5 ) .
this analysis revealed that relative to 
 the melting temperature ( tm ) of the complex - type 
 glycoform , the other glycoforms exhibited reduced tm values : man9glcnac2 ( tm = 2.8  0.7 c ) , hybrid - type glycoforms 
 ( tm = 4.0  0.7 c ) , 
 and endoglycosylated - treated fc ( 5.2  1.0 c ) . 
 therefore , in addition to reducing glycan accessibility to glycosyltransferases , 
 the enzymatic action of golgi -mannosidase ii , to produce complex - type 
 glycans , is permissive for the biosynthesis of a more thermally stabilized 
 fc structure .
we sought to investigate the structural basis for this 
 observation by x - ray crystallography .
hplc analysis of fluorescently labeled 
 n - linked glycans from recombinant 
 igg1expressed in hek 293 t cells in ( a ) the presence and ( b ) the absence 
 of the gnt i inhibitor , swainsonine .
the lower panels show the spectra 
 of glycans following digestion with arthrobacter ureafaciens sialidase .
single 
 thermostability 
 measurements of oligomannose ( man9glcnac2 ) , 
 hybrid , and complex - type glycoforms are shown .
crystallographic structures of igg fc have been reported for a 
 number of glycoforms ranging from oligomannose man9glcnac2 , to homogeneous complex - type , and endoglycosidase - deactivated 
 and aglycosylated states . however , no crystallographic 
 information is available for the hybrid - type fc glycoform that represents 
 the biosynthetic transition point between oligomannose and complex - type 
 glycosylation states .
the hybrid - type 
 glycoform crystallized in the primitive orthorhombic spacegroup , p212121 , with one homodimer 
 in the asymmetric unit .
the c3 protomers were very similar 
 in structure ( 0.3  root - mean - square deviation over 103 equivalent 
 c residues ) and exhibited noncrystallographic 2-fold rotational 
 symmetry .
however , the orientations of the c2 protomers were 
 arranged such that they introduced asymmetry to the homodimer , as 
 has been frequently observed ( figure 6a , b ) .
the protein and carbohydrate components of the c2 
 domain of one chain ( referred to here as chain a ) were largely ordered , 
 while those from the other ( chain b ) exhibited higher b - values where some c3-distal loop regions and associated glycan 
 residues were disordered and not clearly visible in the electron density 
 ( figure 6a ) .
i(hkl)|/hklii(hkl;i ) , where i(hkl;i ) is the intensity 
 of an individual measurement and i(hkl) is the average intensity from multiple observations . rwork = hkl||fobs|  k|fcalc||/hkl|fobs| .
rfree is calculated as for rwork , but using 
 only 5% of the data , which were sequestered prior to refinement .
( a ) the fc structure with the protein moiety shown as a gray ribbon 
 with the n - linked glycan of n297 shown as sticks .
carbohydrate residues 
 are colored and labeled as in figure 1 . a maximum 
 likelihood
weighted 2fo  fc electron density map is plotted around the 
 glycan at 1. ( b ) panel a rotated 70 with a close - up view 
 of the protein and carbohydrate components of the c2 domain 
 from chain a of the hybrid fc crystal structure .
( c ) 
 overlay of the protein backbone of the hybrid ( gray ) , oligomannose 
 ( cyan ; pdb accession code 2wah ) , and complex - type ( pink ; pdb accession code 3ave ) glycoforms .
( d ) 
 the overlay in panel c rotated 90 with a dashed line corresponding 
 to a 17  distance between equivalent ser298 c atoms in 
 the hybrid- and complex - type glycoforms .
the different configurations of the c2 domains 
 may arise 
 due to differences in stabilizing crystallographic packing interactions . 

this analysis reveals that the c2 domain 
 of the more ordered chain a exhibits 37% more buried surface area 
 with symmetry - related molecules than does the corresponding domain 
 in chain b. in chain a of our hybrid - type 
 glycoform , we observe electron density for eight saccharide residues , 
 corresponding to the entirety of the fucosylated oligomannose component 
 of the hybrid - type glycan ( figure 6b ) .
the 3-arm is entirely solvent exposed with the nearest component 
 of a symmetry - related molecule located 9  away . while we can not 
 formally exclude the possibility of selective crystallization of a 
 subset of hybrid - type glycoforms , this distance , and the large accessible 
 volume surrounding the 3-arm , provides no evidence for such a phenomenon . from the observation that the conformations of complex - type glycans 
 are not influenced by the presence of fucose
, we suggest that the structure of the man5glcnac2 component of the hybrid - type glycan reported herein is likely to 
 resemble the man5glcnac2 glycoform that occurs 
 in the preceding stage of the pathway ( figure 1 ) .
this assertion , combined with analysis of the previously reported 
 man9glcnac2 fc structure and the series of complex - type structures by krapp et al .
, now enables us to propose a model of glycan 
 maturation during antibody biogenesis .
the folded man9glcnac2 glycoform is generated 
 following the hydrolysis of the glucose cap and consequent release 
 from the calnexin / calreticulin folding check - point .
all available structures of fc glycoforms show a conserved 
 mode of interaction of the saccharide residues proximal to the protein 
 attachment site .
the man14glcnac14glcnac 
 core and the observable 3-arm residues exhibit highly similar conformations 
 and glycan  protein packing interactions .
in contrast , the 6-arm 
 residues exhibit divergent conformations reflecting the different 
 chemical compositions and/or surrounding environments of the glycan 
 residues ( figure 7 ) .
the structural rearrangements 
 that occur within the oligosaccharide when carbohydrates are modified 
 are illustrated by the hydrolysis of terminal 12 mannose 
 residues of the man9glcnac2 glycoform .
the mand3 residue in the man9glcnac2 structure 
 anchors the oligosaccharide chain to the protein surface at the junction 
 of the c2 and c3 domains ( figure 7a ) . as the solvent accessible mand1 and
mand2 residues are disordered in the crystal structure , hydrolysis of mand3 is likely to induce the 
 observed rearrangement of the 6-arm and cause the 6  shift of 
 manb ( figure 7a , b ) .
this cleavage 
 also results in the associated relaxation of the man416man3 linkage ( from  
 = 69 ,  = 178 ,  = 62 to  
 = 77 ,  = 101 ,  = 30 ; figure 7a , b ) . despite this movement ,
the man4 residue in the man5glcnac2 glycoform 
 is orientated in a direction opposite to that of complex - type structures 
 ( figure 7c ) .
this orientation is maintained 
 by the presence of mana and manb residues 
 of the 6-arm that sterically prevent further rotation of the man416man3 linkage ( figure 7b ) .
the action of gnt i on man5glcnac2 catalyzes 
 the formation of hybrid - type glycans and allows downstream carbohydrate 
 processing ( figure 1 ) .
gnt i transfers 12-linked 
 glcnac to the man4 residue of the 3-arm to form the glcnac512man4 linkage ( figure 7b , c ) . as for many structures of complex - type glycoforms
, 
 we do not observe interpretable electron density for the solvated 
 and mobile residues on this arm .
additionally , gnt i catalysis also 
 renders the core glcnac residue ( glcnac1 ) of the hybrid - type 
 glycan susceptible to 16-fucosylation .
we observe electron density for the fucose and note the 
 conformation closely resembles that of the complex - type glycoform 
 ( figure 7b , c ) .
this supports our assertion 
 that man5glcnac2 glycans are not affected structurally 
 by fucosylation .
golgi -mannosidase 
 ii hydrolyses the 13-linked mana and 16-linked 
 manb residues from the 6-arm and is dependent upon the 
 prior activity of gnt i ( figure 1 ) .
elimination 
 of these residues relieves steric restraints around the 6-arm and 
 allows the reorientation of the man416man3 linkage ( from  = 77 ,  = 101 ,  
 = 30 to  = 62  = 171 ,  = 175 ; 
 figure 7b , c ) , causing close alignment of the 
 glycan to the protein surface .
structural transitions of igg1 fc glycans 
 between ( a ) oligomannose - type 
 ( pdb accession code 2wah ) , ( b ) hybrid - type , and ( c ) complex - type glycosylation ( pdb accession 
 code 3ave ) , 
 as observed by x - ray crystallography .
the central column 
 shows a view with the 6-arm in the foreground . the right - hand column 
 is a close - up and shows the conformational changes occurring within 
 the 6-arm
; an asterix indicates the location of the c6 carbon of the 
 man4 residue .
the protein surface is colored 
 gray , the f243 side - chain is colored pink , and the glycan is colored 
 as in figure 1 except in the close - up where 
 the oxygen atoms of mannose residues are shown in red . following this rearrangement , gnt ii catalyzes 
 the transfer of 
 12-linked glcnac to man4 , allowing 
 the formation of hydrophobic stacking interactions between glcnac5 and phe243 ( figure 7c ) .
the 
 formation of these canonical glycan  protein interactions is 
 consistent with the increased stability ( figure 5 and supporting 
 information figure s1 ) and decreased enzymatic processing of 
 the mature complex - type glycoform relative to the artificially trapped 
 hybrid - type glycoform ( figures 1 , 2 , and 4 ) .
limited downstream compositional 
 heterogeneity of the complex - type glycoform subsequently arises from 
 the partial transfer of galactose to terminal glcnac5 and glcnac5 residues and leads to little change with respect to carbohydrate conformation 
 or thermal stability .
evidence for the suppression 
 of galactosylation and sialylation 
 by the interaction between glcnac5 and phe243 
 is provided by the analysis of igg from a patient with a homozygous 
 mutation in the mgat2 gene.mgat2 encodes gnt ii that catalyzes the transfer 
 of the 6-arm glcnac5 to man4. 
 the igg fc glycans isolated from the patient lacked 6-arm glcnac5 but exhibited significantly elevated 3-arm processing 
 as compared to wild - type structures with the majority of glycans containing 
 the neunac726gal614glcnac5 motif . together with our structural 
 observations , we suggest that the action of golgi -mannosidase 
 ii and gnt ii enhance the glycan  protein interface and limit 
 glycosyltransferase accessibility to the 3-arm .
the use of glycosidase inhibitors 
 and cell - lines with genetically 
 modified glycan processing enzymes offers a powerful route to the 
 isolation of glycoproteins with defined glycan structures .
these methods offer an attractive alternative to direct chemical 
 synthesis and can be readily combined with chemoenzmatic methodologies .
analysis of isolated biosynthetic 
 intermediates of igg1 fc revealed 
 distinct differences in the susceptibility of discrete glycan states 
 to glycosyltransferases .
specifically , we have shown by mass spectrometry 
 that the trapped hybrid - type glycans are more readily accessible to 
 galactosyl and sialyltransferases than are complex - type structures . 

the generation of hybrid - type glycoforms with increased fc sialylation 
 is of note given the enhanced anti - inflammatory functionality exhibited 
 by sialylated fc in , for example , intravenous immunoglobulin therapy .
the biosynthetic intermediates also exhibited 
 reduced stability , 
 an important parameter in the development of antibody therapeutics . through our x - ray crystallographic analysis , 
 we correlate this stability to structural transitions that occur during 
 antibody biogenesis .
we offer a molecular - level explanation for how 
 stability arises from rearrangements of the glycan 
we deduce that glycan - dependent stabilization occurs during 
 golgi -mannosidase ii and gnt ii processing , which respectively 
 cause the relaxation of the 6-arm toward the protein surface and the 
 formation of hydrophobic glycan  protein interfaces .
given 
 the promising portfolio of effector functions exhibited by 
 igg bearing oligomannose and hybrid - type glycans , knowledge of their three - dimensional structure and 
 defined molecular transitions provides a template to support structure - guided 
 stabilization and optimization for the clinic .
j00228 ) was cloned 
 into the phlsec vector and transiently 
 expressed in hek 293 t cells ( atcc number crl-1573 ) , gnt i - deficient 
 hek 293s cells , and in the presence of 
 mannosidase inhibitors to isolate glycoforms of distinct composition .
transfections were performed using 2 mg of dna 
 per liter cell culture medium as previously described .
fc bearing man9glcnac2 and hybrid - type 
 glycosylation were obtained by expression in the presence of 20 and 
 10 m of the inhibitors , kifunensine and swainsonine , respectively ( toronto 
 research chemicals , canada ) .
inhibitors were added at the time of 
 transfection , and the supernatants were harvested after 5 days .
igg1 
 fc glycoproteins were purified at room temperature by immobilized 
 metal - affinity chromatography ( ge healthcare , bucks , uk ) and size 
 exclusion chromatography using a superdex 200 10/30 column ( amersham , 
 bucks , uk ) , in a buffer containing 150 mm nacl and 10 mm tris ph 8.0 . 

plasmids encoding igg1 b12 light and heavy chains were kindly provided 
 by professor dennis burton ( the scripps research institute , ca ) .
the 
 heavy and light chains were transiently cotransfected in hek 293 t 
 cells in the presence or absence of 10 m swainsonine .
igg1 b12 was purified by incubation for 2 h 
 with protein a sepharose ( ge healthcare , uk ) at room temperature . 

the beads were washed with phosphate buffered saline ( pbs ) before 
 elution using 0.1 m citric acid ph 3.4 followed by neutralization 
 and size exclusion chromatography .
the thermal 
 stability of different fc glycoforms was assessed by differential 
 scanning fluorimetry using a stratagene rt pcr 305 instrument .
thermally 
 induced unfolding of purified fc glycoforms was monitored , in triplicate , 
 by measuring absorbance at 610 nm at 1.5 c increments in the 
 presence of sypro orange ( invitrogen , paisley , uk ) , a fluorescent 
 stain sensitive to hydrophobic environments .
oligosaccharides were released from 
 target glycoproteins with peptide - n - glycosidase f 
 ( new englands biolabs ) from coomassie blue - stained nupage gels . excised bands were washed five times alternatively 
 with acetonitrile and deionized water , and rehydrated with a 3000 
 units / ml of aqueous pngase f solution .
after 12 h incubation at 37 
 c , the enzymatically released n - linked glycans were eluted with 
 water .
samples were analyzed by maldi - tof ms with a shimadzu axima 
 tof maldi tof / tof mass spectrometer ( kratos 
 analytical , manchester , uk ) fitted with delayed extraction and a nitrogen 
 laser ( 337 nm ) .
samples were cleaned on a nafion 117 membrane ( aldrich ) , 
 and then prepared for maldi - ms by adding 0.5 l of an aqueous 
 solution of the glycans to the matrix solution ( 0.3 l of a 
 solution of 2,5-dihydroxybenzoic acid in acetonitrile : water ; 1:1 , 
 v : v ) on the stainless steel target plate and allowing it to dry at 
 room temperature .
ms was performed with 
 a waters synapt g2 traveling wave ion mobility mass spectrometer ( waters 
 ms - technologies , manchester , uk ) . samples 
 were dissolved in a solution of methanol : water ( 1:1 , v : v ) containing 
 0.5 m ammonium phosphate and introduced into the instrument with waters 
 thin - wall nanospray capillaries .
the esi capillary voltage was 1.2 
 kv , the cone voltage was 20180 v , and the ion source temperature 
 was 120 c .
the t - wave velocity and peak height voltages were 
 450 m / s and 40 v , respectively .
the t - wave mobility cell contained 
 nitrogen and was operated at a pressure of 0.55 mbar and was used 
 to provide an additional selection stage for the fragmentation experiments . 

fragmentation was performed after mobility separation in the transfer 
 cell with a 3 mass unit selection window and with argon as the collision 
 gas .
the instrument was externally calibrated with sialylated n - glycans 
 released from bovine fetuin .
data acquisition and processing were 
 carried out using waters driftscope ( version 2.1 ) software and masslynx 
 ( version 4.1 ) .
fluorescent labeling of glycans with 2-aminobenzoic 
 acid ( 2-aa ) 
 and subsequent hplc analysis was performed as previously described .
excess 2-aa was removed using a spe - ed amide-2 column ( systematic 
 instruments , uk ) .
hplc was carried out at room temperature in a 20 
 mm ammonium hydroxide solution ( ph 3.9 ) with a linear gradient of 
 acetonitrile and water .
crystals 
 of igg1 fc bearing hybrid - type glycans were grown by sitting - drop 
 vapor diffusion using 100 nl of protein solution ( 11.4 mg ml ) plus 100 nl of precipitant using the previously described semiautomated 
 robotics of the oxford protein production facility .
crystals of igg fc bearing hybrid - type glycans grew at 
 20 c in 20% w / v peg 3350 ph 5.5 with 0.2 m sodium / potassium 
 phosphate after 4 days .
crystals were flash frozen by immersion in 
 a cryoprotectant containing 25% ethylene glycol and transferred to 
 a gaseous nitrogen stream at 100 k. data were collected at beamline 
 bm14 at the european radiation synchrotron facility , grenoble , france 
 ( table 1 ) .
diffraction data were integrated 
 and scaled using the programs denzo and scalepack , and the structure was solved by molecular replacement using 
 the program phaser with the protein chain 
 from a natively glycosylated igg fc ( pdb 
 accession code 3ave ) as a search model .
generally , 
 the hybrid fc structure was refined using refmac5 in the ccp4 suite and included iterative restrained refinement 
 with translation libration screw parametrization and incorporation 
 of automatically generated local noncrystallographic symmetry restraints .
the molecular graphics program coot was used for manual rebuilding , and molprobity was used to validate the model . data processing , refinement , and structure validation 
 statistics are presented in table 1 . throughout this work ,
we have adopted 
 the system of vliegenhart et al . for 
 labeling residues within oligomannose- and biantennary - type oligosaccharides 
 with the additional modifications of 7 and 7 for sialic acid , 1 for 16-linked 
 core fucose ( figure 1 ) .
the symbolic representation of glycans follows that of harvey et al . with residues in both the schematic diagrams 
 and the molecular graphics following the color scheme of the centre 
 for functional glycomics .
carbohydrate fragmentation ions are labeled 
 using the domon and costello nomenclature with an extension by harvey .
dihedral angles were defined using the  x  1  system for crystallography :  = o5c1o  c(x) and  = c1o  c(x)c(x 
1) for man12man 
 and man13man , where x = 2 or 3 , respectively ; 
  = o5c1o  c6 ,  = c1o  c6c5 , 
 and  = o 
 human igg fc glycosylation modulates immunological effector 
 functions 
 such as antibody - dependent cellular cytotoxicity and phagocytosis . 
 
 engineering of fc glycans therefore enables fine - tuning of the therapeutic 
 properties of monoclonal antibodies . 
 the n - linked glycans of fc are 
 typically complex - type , forming a network of noncovalent interactions 
 along the protein surface of the c2 domain . here , we manipulate 
 the mammalian glycan - processing pathway to trap igg1 fc at sequential 
 stages of maturation , from oligomannose- to hybrid- to complex - type 
 glycans , and show that the fc is structurally stabilized following 
 the transition of glycans from their hybrid- to complex - type state . 
 
 x - ray crystallographic analysis of this hybrid - type intermediate reveals 
 that n - linked glycans undergo conformational changes upon maturation , 
 including a flip within the trimannosyl core . 
 our crystal structure 
 of this intermediate reveals a molecular basis for antibody biogenesis 
 and provides a template for the structure - guided engineering of the 
 protein  glycan interface of therapeutic antibodies . 
Introduction
Results and Discussion
Summary and Conclusions
Experimental Procedures