BACKGROUND
the peptides used for intercellular communication are generally short-lived, with half-lives of only a few minutes.
the constant region of human immunoglobulin  is long-lived in the circulation  and has been used to prolong the half-lives of many proteins  <cit> .
many bioactive peptides terminate with an essential c-terminal α-amide  <cit> .
c-terminal α-amidation requires the α-hydroxylation of a peptidylglycine precursor by peptidylglycine α-hydroxylating monooxygenase  and subsequent cleavage of the α-hydroxylated intermediate by peptidyl-α-hydroxyglycine α-amidating lyase  to produce the amidated product and glyoxylate.
a bifunctional type  <dig> integral membrane protein, peptidylglycine α-amidating monooxygenase , is the only enzyme known to catalyze this reaction  <cit> .
we wanted to determine whether fc-amidated peptide fusion proteins could be produced from fc-peptidylglycine precursors and secreted efficiently and whether the products would be biologically active.
chinese hamster ovary  cells are widely used for the production of recombinant antibodies and up to 70 % of all biopharmaceuticals  <cit> .
cho cells were also used to produce phm and pal for crystallographic studies  <cit> .
the cho cell genome encodes pam splice variants similar to those found in rat and human .
endogenous pam activity is detectable in cho cells, but levels are substantially below those found in professional secretory cells such as pituitary endocrine cells and atrial myocytes  <cit> .
consistent with the expression of pam in cho cells, many cho cell monoclonal antibody preparations consist of mixtures of α-amidated and non-amidated product .
we compared the ability of wildtype cho cells and cho cells expressing exogenous membrane or soluble pam to secrete fc-amidated peptide fusion proteins.
the growth hormone signal sequence was placed before the sequence of human fc and a cleavable linker was placed between the c-terminus of fc and the peptidylglycine substrate to facilitate product analysis.
three peptidylglycine substrates were selected for study.
peptide yy , which plays an important role in the response of the endocrine pancreas to oral glucose, terminates with -tyr-nh2; amidation of its c-terminus is essential for bioactivity  <cit> .
neuromedin u , which is expressed in the gastrointestinal tract and brain and plays a major role in regulating energy balance, terminates with -asn-nh2; amidation of its c-terminus is also essential for full bioactivity  <cit> .
glucagon-like peptide  <dig>  which stimulates pancreatic β cells to proliferate and release insulin in response to oral glucose, terminates with -arg-nh2; its c-terminus is not essential for bioactivity  <cit> , but the high km values for –arg-gly peptides suggested that glp1-gly would be a good test case  <cit> .
by placing the peptidylglycine substrate at the c-terminus of the fc-fusion protein, any need for endo- or exoproteases in the biosynthetic pathway was eliminated.
RESULTS
design of vectors encoding fc-amidated peptide precursors
the active site of phm requires the presence of a c-terminal gly; for most neuroendocrine peptide precursors, cleavage by an endoprotease followed by an exoprotease is required to generate the peptidylglycine substrate.
to focus on the requirements for peptide amidation in the cellular environment, we appended the peptidylglycine substrates to the c-terminus of human fc .
the human growth hormone signal was used to ensure efficient protein entry into the secretory pathway.
use of two different linker peptides , was explored and a proteolytic cleavage site was inserted after the linker region so that the peptide product could easily be separated from the fc-region, facilitating analysis of its amidation.
although phm can accommodate any amino acid in the penultimate position, hydrophobic residues facilitate substrate binding  <cit> .
to evaluate the effect of the penultimate amino acid on extent of amidation , we constructed vectors encoding glp1-gly , nmu-gly  and pyy-gly  .fig.
<dig> design of vectors encoding fc-peptidylglycine precursors.
a key features of the fc-peptidylglycine precursors are illustrated.
b the amino acid sequences of human glp1-gly, pyy-gly and nmu-gly are shown .
c eadie-hofstee plots illustrating the effects of synthetic pyy-gly and glp1-gly on conversion of -acetyl-tyr-val-gly into -acetyl-try-val-nh <dig> by purified pam 820 s .
d calculated kinetic parameters for pyy-gly and glp1-gly.
e peak rapid cells were transiently transfected with vectors encoding fc-gs-glp <dig> , fc-ap-glp <dig> , fc-gs-pyy  and fc-ap-pyy ; control non-transfected cells  were analyzed at the same time.
aliquots of spent medium  were fractionated by sds-page and visualized using antibody to human fc; 18 ng human igg  was analyzed at the same time
the ability of glp1-gly and pyy-gly to interact with pam was assessed using synthetic peptides and purified soluble recombinant pam .
pam activity was quantified using -acetyl-tyr-val-gly.
the ability of synthetic glp1-gly and synthetic pyy-gly to inhibit this reaction was assessed; data were plotted in eadie-hofstee format .
while pyy-gly had a ki in the low μm range, the ki of glp1-gly was about 50-fold higher.
predicted vmax values were also much higher for glp1-gly than for pyy-gly, so that their catalytic efficiencies  were similar .
transient expression of the fc-peptidylglycine fusion proteins was used to verify secretion of fc proteins of the expected mass; secreted fusion proteins were detected using antibody to human fc .
spent medium contained only the intact fusion proteins; there was no indication of degradation.
the choice of linker had no effect on secretion of product, but the slightly greater mass of the  <dig> linker  was apparent.
cho lines used to express fc-peptidylglycine fusion proteins
earlier experiments documented the presence of phm activity in cho cell lysates  <cit>  and the secretion of amidated peptides by cho cells expressing exogenous peptide precursors  <cit> .
however, the properties of cho cell pam have not been reported.
analysis of the cricetulus griseus genome revealed the existence of transcripts corresponding to the major pam splice variants detected in rat and human .
while cricetulus griseus isoforms  <dig>   <dig> and  <dig> would be integral membrane proteins, isoforms  <dig> and  <dig> would be soluble proteins.
isoforms  <dig> and  <dig> are products of alternative splicing at exon/intron junctions preceding and immediately following the transmembrane domain and differ in length by only three amino acids.fig.
<dig> characterization of endogenous cho cell pam.
a five isoforms of cricetulus griseus pam are included in the primary refseq assembly of the chinese hamster genome; these isoforms closely resemble those observed in rat, mouse and human: xp_ <dig> , isoform 1; ere <dig> , isoform 2; xp_ <dig> , isoform 3; xp_ <dig> , isoform 4; xp_ <dig> , isoform  <dig>  b the specific activity of endogenous phm in the soluble  and solubilized  crude particulate fractions is indicated.
c the solubilized particulate fraction from two separate preparations of wildtype cho cells  and from cho cells stably expressing pam <dig>  were immunoprecipitated using an affinity-purified antibody to the c-terminus of pam, eluted and fractionated by sds-page, transferred to pvdf membranes and visualized using affinity-purified antibody to the phm domain ; molecular weights of marker proteins analyzed along with pam <dig> are indicated.
a lighter exposure is shown for the other wt sample so that the doublet of  <dig> and 97 kda bands is visible.
apparent molecular weights calculated from three separate analyses are shown ± std dev.
d phm and pal assays were performed on lysates prepared from wildtype cho cells and from cho cells stably expressing rat pam <dig> or rat pam 820 s; lysates were prepared in 1 % tx-100
endogenous phm activity was detected in both crude particulate and soluble fractions prepared from cho cells .
phm specific activity in the particulate fraction was  <dig>  times higher than in the soluble fraction; 70 ± 5 % of the phm activity in cho cell lysates was particulate.
the specific activity of phm in cho cell lysates was below levels observed in corticotrope tumor cells  <cit> , making it impossible to characterize in crude extracts using existing antisera.
immunoprecipitation was used to enrich pam found in the tx- <dig> solubilized particulate fractions prepared from cho cells .
an affinity-purified antibody specific to the c-terminus of pam, which is identical in mouse and chinese hamster, was used to enrich cho cell pam.
after separation by sds-page, antibody specific for the phm region was used to determine the molecular weights of any cross-reactive proteins.
for comparison, a pam <dig> cho cell lysate was immunoprecipitated at the same time.
the minor 113 ± 4 kda cho cell protein recognized by the phm antibody was also visualized by an antibody specific for exon  <dig>  and has the properties expected of isoforms  <dig> and  <dig> .
the closely spaced doublet of bands at 97 ± 3 kda and 92 ± 2 kda could represent chinese hamster isoforms  <dig>   <dig>  and 5; the lower band in the doublet is recognized by the exon  <dig> antibody.
the 77 ± 2 kda pam protein is smaller than any of the characterized splice variants and may represent a cleavage product.
we next compared the level of phm activity in wildtype cho cells to the level present in cho cells stably expressing pam <dig> or pam820s .
based on phm specific activity, expression of pam <dig> and pam820s in the cho lines used for expression of fc-peptidylglycine fusion proteins was 10- and 100-fold higher, respectively, than endogenous phm levels.
in each line, pal specific activity was approximately 10-fold higher than phm specific activity, as expected  <cit> .
characterization of fc-peptidylglycine fusion proteins expressed by wildtype and pam-expressing cho cells
we first examined the ability of stably transfected cho cell lines to produce and secrete the three fc-peptide fusion proteins; the amount of product recovered in spent medium was compared to the amount present in cell extracts .
fc content  and secretion rate  were quantified for each cell line.
for fc-glp <dig> and fc-nmu, fusion proteins containing both the ap and gs linkers were examined; no linker-related differences were apparent.
for fc-pyy, only the fusion protein with a gs linker was examined.
cell content of fc was similar  for all of the lines examined .
the secretion rates varied from 5 % - 20 % of cell content per hour .fig.
<dig> characterization of fc-fusion protein expression and secretion in wildtype cho lines.
samples of cell extract  and spent medium  prepared from cho lines expressing fc-ap-glp <dig>  , fc-gs-pyy  [10 μg  of ce;  <dig>  % of spent medium) and fc-ap-nmu  [10 μg  of ce;  <dig>  % of spent medium) were fractionated by sds-page; samples and a human igg standard were visualized using antibody to human fc.
d using genetools, data from several similar analyses  were quantified to determine μg fc/mg cell protein.
e the secretion rate for fc was calculated for several experiments by quantifying the amount of fc recovered from the cell extract  and from the medium using genetools; for each cell line, secretion rate was expressed as % cell content secreted per hour
the same fc-peptidylglycine fusion proteins were also expressed in cho lines expressing exogenous pam.
fc content and secretion rate were quantified for the pam-expressing cho cell lines in the same manner as for wildtype cho cell lines .
fc-glp <dig> with the ap linker and fc-pyy with gs linker were expressed in pam <dig> cho cells.
fc-nmu with both the ap and gs linkers was expressed in pam820s cho cells.
western blot analyses are shown , along with quantification of fc content  and secretion rate .
as for wildtype cho lines, cell content of fc ranged from 2–4 μg fc/mg cell protein for all of the lines and fc secretion rates were all in the range of 12-22 % of cell content per hour.fig.
<dig> characterization of fc-fusion protein expression in cho lines expressing pam.
fc-ap-glp <dig>  and fc-gs-pyy  were expressed in pam <dig> cho cells; fc-ap-nmu  was expressed in pam820s cho cells.
aliquots of cell extract  and spent medium  were prepared as described for fig.
<dig>  d using genetools, data from several similar analyses were quantified to determine μg fc/mg cell protein  and fc secretion rate
purification of secreted fc-fusion proteins and evaluation of amidation status
monolayer cultures of stably transfected cho lines were fed with complete serum-free medium every other day for a week; after concentration, spent media were applied to protein a cartridges and bound fc-fusion proteins were eluted with low ph buffer.
sds-page analysis of the eluates, along with recombinant fc, established their purity .
yields ranged from  <dig> to 5 mg purified fc-fusion protein per liter of spent medium.
intact mass analysis of each product revealed the presence of a disulfide bonded dimer with a biantennary, core-fucosylated glycan.
the observed glycosylation heterogeneity illustrated for the fc-ap-glp <dig> construct  was more diverse than is typically encountered in manufacturing cho cell lines used for biotherapeutic  production, as exemplified by the fc standard .
the presence of some hybrid glycoforms would indicate that a low percentage of the fc-fusion proteins were secreted prematurely  or there was competition for substrate which interfered with glycosyltransferases.fig.
<dig> purification and mass spectroscopic analysis of fc-fusion proteins.
a after concentration using a vivaflow flip flow filtration system and a centricon, secreted fc-fusion proteins were purified by binding to hitrap protein a cartridges; bound protein was eluted by application of low ph buffer and aliquots  were subjected to sds-page; purified fc was analyzed at the same time.
comparison of glycan heterogeneity by intact mass analysis of fc-ap-glp <dig>  and fc from manufacturing cell line .
peak labels are shorthand notation for glycosylation state.
all molecules exhibited two bi-antennary core fucosylated glycans  with an additional gn  galactose and sn  sialic acid  moieties distributed between the two glycans
intact mass analysis was not precise enough to determine the degree of c-terminal amidation.
therefore, extent of amidation was quantified by analysis of the hrv3c rhinovirus protease cleavage products .
peptidylglycine precursors differ from their amidated products by a mass of +58; amidated peptides differ from the corresponding peptide with a free cooh-terminus by a mass of − <dig> and a charge change  at neutral ph.
intact mass analysis of the cleaved proteins allowed reliable identification of amidated product and glycine-extended precursor .
product lacking the cooh-terminal gly, with a free cooh-terminus was not identified.
some off-target cleavage by rhinovirus protease was observed; instead of the expected cleavage between q and g <dig> , cleavage occasionally occurred before f <dig> or q <dig>  each purified product was analyzed in triplicate.
for each substrate, the percent amidated was calculated by summing amidated plus glycine-extended peptide to determine the total amount of peptide recovered, and assumes there is no difference in hrv3c specificity or cleavage rate between the two substrates.fig.
<dig> reversed-phase hplc-ms analysis of hrv3c cleavage products.
a total ion chromatogram ; b single-charge deconvoluted mass spectrum  from the m/z data under the peptide peak.
c purified fc-fusion proteins were cleaved with hrv3c protease and subjected to mass spectroscopic analysis as described in methods.
each sample was analyzed in triplicate; standard errors are shown
fc-glp <dig> was almost 90 % amidated, even when expressed in wildtype cho cells having only endogenous levels of pam .
no difference in extent of fc-glp <dig> amidation was observed in cho cells expressing pam <dig>  in contrast, less than 30 % of the fc-pyy produced by wildtype cho cells was amidated; expression of pam <dig> increased the extent of pyy amidation by about a factor of two .
approximately 71 % of the fc-nmu produced by wildtype cho cells was amidated; fc-ap-nmu and fc-gs-nmu did not differ in their extent of amidation.
fc-nmu produced by cho cells expressing soluble pam  was over 90 % amidated, a significant increase over the extent of amidation observed in wildtype cho cells.
assessment of biological activity
the peptidylglycine substrates tested each yield amidated peptides that interact with specific gpcrs.
we therefore used cell-based receptor assays to test the biological activity of two of the purified secreted fc-fusion proteins.
the concentration of each fc-fusion protein was determined by comparison to a standard curve generated using purified human fc analyzed on the same western blot.
the bioassay for glp <dig> used hek cells expressing the glp-1r; synthetic glp1-ser <dig> was used as the standard and had an ec <dig> of  <dig>  pm .
the nh2-terminus of glp <dig> plays an essential role in its interaction with receptor and the addition of the gp dipeptide to the nh2-terminus of synthetic glp <dig> increased its ec <dig> to  <dig>  pm .
the corresponding glycine-extended peptide had a similar ec <dig>  as expected for a ligand/receptor interaction that is known not to require the cooh-terminus of the ligand  <cit> .
although the intact fc-glp <dig> fusion proteins tested yielded dose response curves that were parallel to those of glp <dig>  they were at least  <dig> -fold less potent than glp1; their ec <dig> values were in the  <dig> nm range .fig.
<dig> bioassay of purified fc-glp <dig> fusion proteins.
hek cells expressing the glp-1r were used to assess the ability of fc-glp <dig> fusion proteins purified from the spent medium of different cho cell lines to stimulate camp production.
a the activity of glp <dig>  gp-glp1-nh <dig> and gp-glp1was compared; as expected, amidation did not affect bioactivity while appending the gp dipeptide to its amino-terminus reduced the potency of glp1-nh <dig>  b the activity of glp <dig> was compared to that of fc-ap-glp <dig> fusion proteins purified from the spent media of wt cho cells, cho cells expressing pam <dig> and wt cho cells treated with 10 μm bathocuproine disulfonate to inhibit endogenous phm throughout the entire collection period.
parallel dose–response curves were observed for all of the peptides and proteins tested; fc-glp <dig> was at least  <dig> -fold less active than glp <dig>  the y-axis shows the tr-fret signal at 665 nm; the signal obtained by cells in the absence of any added peptide is indicated
the bioassay for pyy used cho cells expressing the npy-2r; synthetic pyynh <dig> was used as the standard and had an ic <dig> of  <dig>  nm .
synthetic peptides corresponding to full-length pyy with the gp dipeptide left by rhinovirus protease cleavage of the fc-pyy substrate were used to evaluate the effect of cooh-terminal amidation.
gp-pyy-nh <dig> was slightly more potent than pyynh <dig> while gp-pyy-gly was 1000-fold less potent .
the fc-pyy products examined yielded dose response curves that were parallel to those of pyynh <dig> and gp-pyy-nh <dig>  although their ic <dig> values were at least 100-fold higher than the synthetic peptide, the fc-pyy products were 10-fold more potent than gp-pyy-gly.fig.
<dig> bioassay of purified fc-pyy fusion proteins.
cho cells expressing the npy-2r were used to assess the ability of fc-pyy fusion proteins purified from the spent medium of different cho lines to inhibit forskolin-stimulated camp production; pyy binding to npy-2r activates gαi, decreasing camp production.
synthetic pyynh <dig> was more potent than synthetic gp-pyy-nh2; synthetic gp-pyy-gly was at least  <dig> -fold less potent than the corresponding amidated peptide.
although the fc-pyy fusion protein produced by pam <dig> cells was about 200-fold less active than synthetic gp-pyy-nh <dig>  it was 10-fold more active than synthetic gp-pyy-gly.
parallel dose–response curves were observed for all of the peptides and proteins tested
discussion
cho cells have served as excellent mammalian cell factories for making proteins that are difficult or impossible to create in bacterial and yeast systems  <cit> ; the α-amidating enzyme, pam, falls into this category and has been successfully expressed in cho cells .
thus, to produce large, α-amidated proteins for therapeutic or investigational purposes, cho cells were selected.
the plasma half-life of many peptides has been increased several orders of magnitude by fusion with the fc region of human immunoglobulin  <cit> , making the fc-propeptide fusion a desirable starting point to make long-lived proteins terminating with the α-amidated peptide of interest.
although the specific activity of endogenous cho cell pam is low compared to endocrine cells or atrial myocytes  <cit> , it is clearly capable of α-amidating exogenous proteins.
cho cells expressing monoclonal antibodies that terminate with a c-terminal gly residue are often partially α-amidated  <cit> .
the ability of endogenous cho cell pam to α-amidate a significant amount of each fc-peptidylglycine precursor is consistent with the properties of pam established using test tube assays and the levels of enzyme and substrate expressed.
when assayed with substrate levels well below the km of the enzyme , non-transfected cho cells had a specific activity of  <dig>  nmol/mg protein/h ; based on michaelis-menten kinetics and allowing for the molecular weight of the fusion protein, this corresponds to a maximal amidation rate of 45 μg fc-peptide/mg protein/h.
since the cell lines studied here contained only 1–3 μg fc-peptide/mg protein and secreted only  <dig> to 20 % of their cell content per h , they would be expected to α-amidate a significant fraction of the secreted fc-peptide.
cell lines producing significantly more fc-peptide may need exogenous pam and increased storage capacity to produce more extensively amidated product.
comparison of cell-based and test tube assays suggests that pam is operating at close to vmax in cells – its affinity for the fc-peptidylglycine substrate is not a major factor.
glp1-gly, with a penultimate arg residue, was more extensively amidated than pyy, with a penultimate tyr residue .
by comparison, in test tube assays, peptides ending in –aromatic-gly are better substrates than peptides ending in –charged-gly  <cit> .
the more rapid secretion of fc-pyy than fc-glp <dig>  may contribute to this phenomenon, although an explanation for this difference is not apparent.
the very high concentration of protein in the lumen of the secretory pathway  is difficult to mimic in test tube assays  <cit> .
both the cellular machinery and substrate will need to be optimized in order to achieve the maximal yield of extensively α-amidated product.
clearly the rate of secretion of the fc-fusion peptide  has a role, since the fc-peptides secreted slowly  were highly amidated without the need for additional pam .
by contrast, the cells which secreted the fusion peptide rapidly  showed less extensive α-amidation of the peptide  and the extent of amidation was improved by expression of exogenous pam .
fc-pyy provides a better test case for assessing modifications aimed at increasing the extent of α-amidation than fc-glp <dig>
CONCLUSIONS
the ultimate goal of this work is creation of long-lived peptides for therapeutic or investigational uses.
the peptides chosen for this initial study were selected to answer specific questions about the methodology and the ability of pam to handle different substrates.
the limited ability of fc-pyy-nh <dig> to activate the npy-2r may reflect the role of the n-terminal region of pyy in its interaction with receptor  <cit>  or interference by the fc-region with access of pyy-nh <dig> to its receptor.
a different carrier protein might better confer stability to the fusion protein without compromising receptor accessibility.
the properties of the carrier protein may also determine the speed with which it traverse the secretory pathway and the time over which it is exposed to pam.
the properties of pam are unlikely to limit the choice of carrier protein.
fusion protein stability will need to be assessed; the ability of purified fc-pyy to activate the npy-2r may reflect cleavage during the incubation with cells.
it is not currently feasible to attempt to recover the peptide after the bioassay, and any cleaved peptide that was endocytosed by the reporter cells would also be missed.
whether using their endogenous enzyme or exogenous pam, cho cells can efficiently amidate large proteins that terminate with a gly residue.

 the peptides used for intercellular communication are generally short-lived, with half-lives of only a few minutes. the constant region of human immunoglobulin is long-lived in the circulation and has been used to prolong the half-lives of many proteins. many bioactive peptides terminate with an essential c-terminal α-amide. c-terminal α-amidation requires the α-hydroxylation of a peptidylglycine precursor by peptidylglycine α-hydroxylating monooxygenase and subsequent cleavage of the α-hydroxylated intermediate by peptidyl-α-hydroxyglycine α-amidating lyase to produce the amidated product and glyoxylate. a bifunctional type integral membrane protein, peptidylglycine α-amidating monooxygenase, is the only enzyme known to catalyze this reaction. we wanted to determine whether fc-amidated peptide fusion proteins could be produced from fc-peptidylglycine precursors and secreted efficiently and whether the products would be biologically active. chinese hamster ovary cells are widely used for the production of recombinant antibodies and up to 70 % of all biopharmaceuticals. cho cells were also used to produce phm and pal for crystallographic studies. the cho cell genome encodes pam splice variants similar to those found in rat and human. endogenous pam activity is detectable in cho cells, but levels are substantially below those found in professional secretory cells such as pituitary endocrine cells and atrial myocytes. consistent with the expression of pam in cho cells, many cho cell monoclonal antibody preparations consist of mixtures of α-amidated and non-amidated product. we compared the ability of wildtype cho cells and cho cells expressing exogenous membrane or soluble pam to secrete fc-amidated peptide fusion proteins. the growth hormone signal sequence was placed before the sequence of human fc and a cleavable linker was placed between the c-terminus of fc and the peptidylglycine substrate to facilitate product analysis. three peptidylglycine substrates were selected for study. peptide yy, which plays an important role in the response of the endocrine pancreas to oral glucose, terminates with -tyr-nh2; amidation of its c-terminus is essential for bioactivity. neuromedin u, which is expressed in the gastrointestinal tract and brain and plays a major role in regulating energy balance, terminates with -asn-nh2; amidation of its c-terminus is also essential for full bioactivity. glucagon-like peptide which stimulates pancreatic β cells to proliferate and release insulin in response to oral glucose, terminates with -arg-nh2; its c-terminus is not essential for bioactivity, but the high km values for –arg-gly peptides suggested that glp1-gly would be a good test case. by placing the peptidylglycine substrate at the c-terminus of the fc-fusion protein, any need for endo- or exoproteases in the biosynthetic pathway was eliminated.  design of vectors encoding fc-amidated peptide precursors the active site of phm requires the presence of a c-terminal gly; for most neuroendocrine peptide precursors, cleavage by an endoprotease followed by an exoprotease is required to generate the peptidylglycine substrate. to focus on the requirements for peptide amidation in the cellular environment, we appended the peptidylglycine substrates to the c-terminus of human fc. the human growth hormone signal was used to ensure efficient protein entry into the secretory pathway. use of two different linker peptides, was explored and a proteolytic cleavage site was inserted after the linker region so that the peptide product could easily be separated from the fc-region, facilitating analysis of its amidation. although phm can accommodate any amino acid in the penultimate position, hydrophobic residues facilitate substrate binding. to evaluate the effect of the penultimate amino acid on extent of amidation, we constructed vectors encoding glp1-gly, nmu-gly and pyy-gly.fig. design of vectors encoding fc-peptidylglycine precursors. a key features of the fc-peptidylglycine precursors are illustrated. b the amino acid sequences of human glp1-gly, pyy-gly and nmu-gly are shown. c eadie-hofstee plots illustrating the effects of synthetic pyy-gly and glp1-gly on conversion of -acetyl-tyr-val-gly into -acetyl-try-val-nh by purified pam 820 s. d calculated kinetic parameters for pyy-gly and glp1-gly. e peak rapid cells were transiently transfected with vectors encoding fc-gs-glp, fc-ap-glp, fc-gs-pyy and fc-ap-pyy; control non-transfected cells were analyzed at the same time. aliquots of spent medium were fractionated by sds-page and visualized using antibody to human fc; 18 ng human igg was analyzed at the same time the ability of glp1-gly and pyy-gly to interact with pam was assessed using synthetic peptides and purified soluble recombinant pam. pam activity was quantified using -acetyl-tyr-val-gly. the ability of synthetic glp1-gly and synthetic pyy-gly to inhibit this reaction was assessed; data were plotted in eadie-hofstee format. while pyy-gly had a ki in the low μm range, the ki of glp1-gly was about 50-fold higher. predicted vmax values were also much higher for glp1-gly than for pyy-gly, so that their catalytic efficiencies were similar. transient expression of the fc-peptidylglycine fusion proteins was used to verify secretion of fc proteins of the expected mass; secreted fusion proteins were detected using antibody to human fc. spent medium contained only the intact fusion proteins; there was no indication of degradation. the choice of linker had no effect on secretion of product, but the slightly greater mass of the linker was apparent. cho lines used to express fc-peptidylglycine fusion proteins earlier experiments documented the presence of phm activity in cho cell lysates and the secretion of amidated peptides by cho cells expressing exogenous peptide precursors. however, the properties of cho cell pam have not been reported. analysis of the cricetulus griseus genome revealed the existence of transcripts corresponding to the major pam splice variants detected in rat and human. while cricetulus griseus isoforms and would be integral membrane proteins, isoforms and would be soluble proteins. isoforms and are products of alternative splicing at exon/intron junctions preceding and immediately following the transmembrane domain and differ in length by only three amino acids.fig. characterization of endogenous cho cell pam. a five isoforms of cricetulus griseus pam are included in the primary refseq assembly of the chinese hamster genome; these isoforms closely resemble those observed in rat, mouse and human: xp_, isoform 1; ere, isoform 2; xp_, isoform 3; xp_, isoform 4; xp_, isoform b the specific activity of endogenous phm in the soluble and solubilized crude particulate fractions is indicated. c the solubilized particulate fraction from two separate preparations of wildtype cho cells and from cho cells stably expressing pam were immunoprecipitated using an affinity-purified antibody to the c-terminus of pam, eluted and fractionated by sds-page, transferred to pvdf membranes and visualized using affinity-purified antibody to the phm domain; molecular weights of marker proteins analyzed along with pam are indicated. a lighter exposure is shown for the other wt sample so that the doublet of and 97 kda bands is visible. apparent molecular weights calculated from three separate analyses are shown ± std dev. d phm and pal assays were performed on lysates prepared from wildtype cho cells and from cho cells stably expressing rat pam or rat pam 820 s; lysates were prepared in 1 % tx-100 endogenous phm activity was detected in both crude particulate and soluble fractions prepared from cho cells. phm specific activity in the particulate fraction was times higher than in the soluble fraction; 70 ± 5 % of the phm activity in cho cell lysates was particulate. the specific activity of phm in cho cell lysates was below levels observed in corticotrope tumor cells, making it impossible to characterize in crude extracts using existing antisera. immunoprecipitation was used to enrich pam found in the tx- solubilized particulate fractions prepared from cho cells. an affinity-purified antibody specific to the c-terminus of pam, which is identical in mouse and chinese hamster, was used to enrich cho cell pam. after separation by sds-page, antibody specific for the phm region was used to determine the molecular weights of any cross-reactive proteins. for comparison, a pam cho cell lysate was immunoprecipitated at the same time. the minor 113 ± 4 kda cho cell protein recognized by the phm antibody was also visualized by an antibody specific for exon and has the properties expected of isoforms and. the closely spaced doublet of bands at 97 ± 3 kda and 92 ± 2 kda could represent chinese hamster isoforms and 5; the lower band in the doublet is recognized by the exon antibody. the 77 ± 2 kda pam protein is smaller than any of the characterized splice variants and may represent a cleavage product. we next compared the level of phm activity in wildtype cho cells to the level present in cho cells stably expressing pam or pam820s. based on phm specific activity, expression of pam and pam820s in the cho lines used for expression of fc-peptidylglycine fusion proteins was 10- and 100-fold higher, respectively, than endogenous phm levels. in each line, pal specific activity was approximately 10-fold higher than phm specific activity, as expected. characterization of fc-peptidylglycine fusion proteins expressed by wildtype and pam-expressing cho cells we first examined the ability of stably transfected cho cell lines to produce and secrete the three fc-peptide fusion proteins; the amount of product recovered in spent medium was compared to the amount present in cell extracts. fc content and secretion rate were quantified for each cell line. for fc-glp and fc-nmu, fusion proteins containing both the ap and gs linkers were examined; no linker-related differences were apparent. for fc-pyy, only the fusion protein with a gs linker was examined. cell content of fc was similar for all of the lines examined. the secretion rates varied from 5 % - 20 % of cell content per hour.fig. characterization of fc-fusion protein expression and secretion in wildtype cho lines. samples of cell extract and spent medium prepared from cho lines expressing fc-ap-glp, fc-gs-pyy [10 μg of ce;  % of spent medium) and fc-ap-nmu [10 μg of ce;  % of spent medium) were fractionated by sds-page; samples and a human igg standard were visualized using antibody to human fc. d using genetools, data from several similar analyses were quantified to determine μg fc/mg cell protein. e the secretion rate for fc was calculated for several experiments by quantifying the amount of fc recovered from the cell extract and from the medium using genetools; for each cell line, secretion rate was expressed as % cell content secreted per hour the same fc-peptidylglycine fusion proteins were also expressed in cho lines expressing exogenous pam. fc content and secretion rate were quantified for the pam-expressing cho cell lines in the same manner as for wildtype cho cell lines. fc-glp with the ap linker and fc-pyy with gs linker were expressed in pam cho cells. fc-nmu with both the ap and gs linkers was expressed in pam820s cho cells. western blot analyses are shown, along with quantification of fc content and secretion rate. as for wildtype cho lines, cell content of fc ranged from 2–4 μg fc/mg cell protein for all of the lines and fc secretion rates were all in the range of 12-22 % of cell content per hour.fig. characterization of fc-fusion protein expression in cho lines expressing pam. fc-ap-glp and fc-gs-pyy were expressed in pam cho cells; fc-ap-nmu was expressed in pam820s cho cells. aliquots of cell extract and spent medium were prepared as described for fig.  d using genetools, data from several similar analyses were quantified to determine μg fc/mg cell protein and fc secretion rate purification of secreted fc-fusion proteins and evaluation of amidation status monolayer cultures of stably transfected cho lines were fed with complete serum-free medium every other day for a week; after concentration, spent media were applied to protein a cartridges and bound fc-fusion proteins were eluted with low ph buffer. sds-page analysis of the eluates, along with recombinant fc, established their purity. yields ranged from to 5 mg purified fc-fusion protein per liter of spent medium. intact mass analysis of each product revealed the presence of a disulfide bonded dimer with a biantennary, core-fucosylated glycan. the observed glycosylation heterogeneity illustrated for the fc-ap-glp construct was more diverse than is typically encountered in manufacturing cho cell lines used for biotherapeutic production, as exemplified by the fc standard. the presence of some hybrid glycoforms would indicate that a low percentage of the fc-fusion proteins were secreted prematurely or there was competition for substrate which interfered with glycosyltransferases.fig. purification and mass spectroscopic analysis of fc-fusion proteins. a after concentration using a vivaflow flip flow filtration system and a centricon, secreted fc-fusion proteins were purified by binding to hitrap protein a cartridges; bound protein was eluted by application of low ph buffer and aliquots were subjected to sds-page; purified fc was analyzed at the same time. comparison of glycan heterogeneity by intact mass analysis of fc-ap-glp and fc from manufacturing cell line. peak labels are shorthand notation for glycosylation state. all molecules exhibited two bi-antennary core fucosylated glycans with an additional gn galactose and sn sialic acid moieties distributed between the two glycans intact mass analysis was not precise enough to determine the degree of c-terminal amidation. therefore, extent of amidation was quantified by analysis of the hrv3c rhinovirus protease cleavage products. peptidylglycine precursors differ from their amidated products by a mass of +58; amidated peptides differ from the corresponding peptide with a free cooh-terminus by a mass of − and a charge change at neutral ph. intact mass analysis of the cleaved proteins allowed reliable identification of amidated product and glycine-extended precursor. product lacking the cooh-terminal gly, with a free cooh-terminus was not identified. some off-target cleavage by rhinovirus protease was observed; instead of the expected cleavage between q and g, cleavage occasionally occurred before f or q each purified product was analyzed in triplicate. for each substrate, the percent amidated was calculated by summing amidated plus glycine-extended peptide to determine the total amount of peptide recovered, and assumes there is no difference in hrv3c specificity or cleavage rate between the two substrates.fig. reversed-phase hplc-ms analysis of hrv3c cleavage products. a total ion chromatogram; b single-charge deconvoluted mass spectrum from the m/z data under the peptide peak. c purified fc-fusion proteins were cleaved with hrv3c protease and subjected to mass spectroscopic analysis as described in methods. each sample was analyzed in triplicate; standard errors are shown fc-glp was almost 90 % amidated, even when expressed in wildtype cho cells having only endogenous levels of pam. no difference in extent of fc-glp amidation was observed in cho cells expressing pam in contrast, less than 30 % of the fc-pyy produced by wildtype cho cells was amidated; expression of pam increased the extent of pyy amidation by about a factor of two. approximately 71 % of the fc-nmu produced by wildtype cho cells was amidated; fc-ap-nmu and fc-gs-nmu did not differ in their extent of amidation. fc-nmu produced by cho cells expressing soluble pam was over 90 % amidated, a significant increase over the extent of amidation observed in wildtype cho cells. assessment of biological activity the peptidylglycine substrates tested each yield amidated peptides that interact with specific gpcrs. we therefore used cell-based receptor assays to test the biological activity of two of the purified secreted fc-fusion proteins. the concentration of each fc-fusion protein was determined by comparison to a standard curve generated using purified human fc analyzed on the same western blot. the bioassay for glp used hek cells expressing the glp-1r; synthetic glp1-ser was used as the standard and had an ec of pm. the nh2-terminus of glp plays an essential role in its interaction with receptor and the addition of the gp dipeptide to the nh2-terminus of synthetic glp increased its ec to pm. the corresponding glycine-extended peptide had a similar ec as expected for a ligand/receptor interaction that is known not to require the cooh-terminus of the ligand. although the intact fc-glp fusion proteins tested yielded dose response curves that were parallel to those of glp they were at least -fold less potent than glp1; their ec values were in the nm range.fig. bioassay of purified fc-glp fusion proteins. hek cells expressing the glp-1r were used to assess the ability of fc-glp fusion proteins purified from the spent medium of different cho cell lines to stimulate camp production. a the activity of glp gp-glp1-nh and gp-glp1was compared; as expected, amidation did not affect bioactivity while appending the gp dipeptide to its amino-terminus reduced the potency of glp1-nh b the activity of glp was compared to that of fc-ap-glp fusion proteins purified from the spent media of wt cho cells, cho cells expressing pam and wt cho cells treated with 10 μm bathocuproine disulfonate to inhibit endogenous phm throughout the entire collection period. parallel dose–response curves were observed for all of the peptides and proteins tested; fc-glp was at least -fold less active than glp the y-axis shows the tr-fret signal at 665 nm; the signal obtained by cells in the absence of any added peptide is indicated the bioassay for pyy used cho cells expressing the npy-2r; synthetic pyynh was used as the standard and had an ic of nm. synthetic peptides corresponding to full-length pyy with the gp dipeptide left by rhinovirus protease cleavage of the fc-pyy substrate were used to evaluate the effect of cooh-terminal amidation. gp-pyy-nh was slightly more potent than pyynh while gp-pyy-gly was 1000-fold less potent. the fc-pyy products examined yielded dose response curves that were parallel to those of pyynh and gp-pyy-nh although their ic values were at least 100-fold higher than the synthetic peptide, the fc-pyy products were 10-fold more potent than gp-pyy-gly.fig. bioassay of purified fc-pyy fusion proteins. cho cells expressing the npy-2r were used to assess the ability of fc-pyy fusion proteins purified from the spent medium of different cho lines to inhibit forskolin-stimulated camp production; pyy binding to npy-2r activates gαi, decreasing camp production. synthetic pyynh was more potent than synthetic gp-pyy-nh2; synthetic gp-pyy-gly was at least -fold less potent than the corresponding amidated peptide. although the fc-pyy fusion protein produced by pam cells was about 200-fold less active than synthetic gp-pyy-nh it was 10-fold more active than synthetic gp-pyy-gly. parallel dose–response curves were observed for all of the peptides and proteins tested discussion cho cells have served as excellent mammalian cell factories for making proteins that are difficult or impossible to create in bacterial and yeast systems; the α-amidating enzyme, pam, falls into this category and has been successfully expressed in cho cells. thus, to produce large, α-amidated proteins for therapeutic or investigational purposes, cho cells were selected. the plasma half-life of many peptides has been increased several orders of magnitude by fusion with the fc region of human immunoglobulin, making the fc-propeptide fusion a desirable starting point to make long-lived proteins terminating with the α-amidated peptide of interest. although the specific activity of endogenous cho cell pam is low compared to endocrine cells or atrial myocytes, it is clearly capable of α-amidating exogenous proteins. cho cells expressing monoclonal antibodies that terminate with a c-terminal gly residue are often partially α-amidated. the ability of endogenous cho cell pam to α-amidate a significant amount of each fc-peptidylglycine precursor is consistent with the properties of pam established using test tube assays and the levels of enzyme and substrate expressed. when assayed with substrate levels well below the km of the enzyme, non-transfected cho cells had a specific activity of  nmol/mg protein/h; based on michaelis-menten kinetics and allowing for the molecular weight of the fusion protein, this corresponds to a maximal amidation rate of 45 μg fc-peptide/mg protein/h. since the cell lines studied here contained only 1–3 μg fc-peptide/mg protein and secreted only to 20 % of their cell content per h, they would be expected to α-amidate a significant fraction of the secreted fc-peptide. cell lines producing significantly more fc-peptide may need exogenous pam and increased storage capacity to produce more extensively amidated product. comparison of cell-based and test tube assays suggests that pam is operating at close to vmax in cells – its affinity for the fc-peptidylglycine substrate is not a major factor. glp1-gly, with a penultimate arg residue, was more extensively amidated than pyy, with a penultimate tyr residue. by comparison, in test tube assays, peptides ending in –aromatic-gly are better substrates than peptides ending in –charged-gly. the more rapid secretion of fc-pyy than fc-glp may contribute to this phenomenon, although an explanation for this difference is not apparent. the very high concentration of protein in the lumen of the secretory pathway is difficult to mimic in test tube assays. both the cellular machinery and substrate will need to be optimized in order to achieve the maximal yield of extensively α-amidated product. clearly the rate of secretion of the fc-fusion peptide has a role, since the fc-peptides secreted slowly were highly amidated without the need for additional pam. by contrast, the cells which secreted the fusion peptide rapidly showed less extensive α-amidation of the peptide and the extent of amidation was improved by expression of exogenous pam. fc-pyy provides a better test case for assessing modifications aimed at increasing the extent of α-amidation than fc-glp  the ultimate goal of this work is creation of long-lived peptides for therapeutic or investigational uses. the peptides chosen for this initial study were selected to answer specific questions about the methodology and the ability of pam to handle different substrates. the limited ability of fc-pyy-nh to activate the npy-2r may reflect the role of the n-terminal region of pyy in its interaction with receptor or interference by the fc-region with access of pyy-nh to its receptor. a different carrier protein might better confer stability to the fusion protein without compromising receptor accessibility. the properties of the carrier protein may also determine the speed with which it traverse the secretory pathway and the time over which it is exposed to pam. the properties of pam are unlikely to limit the choice of carrier protein. fusion protein stability will need to be assessed; the ability of purified fc-pyy to activate the npy-2r may reflect cleavage during the incubation with cells. it is not currently feasible to attempt to recover the peptide after the bioassay, and any cleaved peptide that was endocytosed by the reporter cells would also be missed. whether using their endogenous enzyme or exogenous pam, cho cells can efficiently amidate large proteins that terminate with a gly residue. 
glucagon-like peptide, peptide yy and neuromedin u were expressed and purified from stable cho lines; since the α-amide group is essential for full biological potency of many peptides, fc-fusion peptides were expressed in cho lines stably expressing the α-amidating enzyme, peptidylglycine α-amidating monooxygenase. amidationcho cellsmass spectrometryglucagon-like peptide 1peptide yyneuromedin uissue-copyright-statement© the author 2015 both problems are potentially solved by producing peptides as fusions with the fc region of human immunoglobulin. purified fusion proteins were analyzed intact and after hrv3c rhinovirus protease cleavage, at a site in the linker separating the fc region from the peptide, by mass spectrometry and amide-specific immunoassays. cho cells express measurable endogenous pam activity, amidating 25 % of fc-pyy and almost 90 % of fc-glp expression of exogenous pam increased the level of peptide amidation to 50 % of fc-pyy and 95 % of fc-nmu. the fc fusions were expressed at 1–  μg/mg cell protein and secreted at 5-20 % of cell content per hour, in a peptide-specific manner.
 the therapeutic use of α-amidated peptides has increased dramatically, but there are major impediments to wider use of such peptides. larger peptides are expensive to synthesize, and short plasma half-lives frequently limit the clinical circumstances in which the peptides would be useful. both problems are potentially solved by producing peptides as fusions with the fc region of human immunoglobulin. methods glucagon-like peptide, peptide yy and neuromedin u were expressed and purified from stable cho lines; since the α-amide group is essential for full biological potency of many peptides, fc-fusion peptides were expressed in cho lines stably expressing the α-amidating enzyme, peptidylglycine α-amidating monooxygenase. purified fusion proteins were analyzed intact and after hrv3c rhinovirus protease cleavage, at a site in the linker separating the fc region from the peptide, by mass spectrometry and amide-specific immunoassays.  the fc fusions were expressed at 1–  μg/mg cell protein and secreted at 5-20 % of cell content per hour, in a peptide-specific manner. cho cells express measurable endogenous pam activity, amidating 25 % of fc-pyy and almost 90 % of fc-glp expression of exogenous pam increased the level of peptide amidation to 50 % of fc-pyy and 95 % of fc-nmu. the fc-glp fusions were -fold less active than synthetic glp in a cell-receptor cyclic amp-based assay, as expected since the amino terminal of glp is essential for full biological activity. the fc-pyy fusions were 100-fold less active than pyy-nh but 10-fold more active than non-amidated pyy-gly.  this type of approach can be used for the production of stabilized α-amidated peptides aimed at clinical trials. keywords amidationcho cellsmass spectrometryglucagon-like peptide 1peptide yyneuromedin uissue-copyright-statement© the author 2015 