The sialylated oligosaccharides of recombinant bovine lutropin modulate hormone bioactivity.

P L Smith,D Kaetzel,J Nilson,J U Baenziger

doi:10.1016/s0021-9258(19)40131-2

P L Smith, D Kaetzel + Show 2 more

Open Access

https://doi.org/10.1016/s0021-9258(19)40131-2

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Abstract

The Asn-linked oligosaccharides from bovine lutropin (bLH(Pit] are predominantly dibranched complex-type structures with the terminal sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha. Recombinant bLH expressed in Chinese hamster ovary cells (bLH(CHO] bears di- (60%) and tribranched (30%) complex-type oligosaccharides; however, these terminate in the sequence Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,2Man alpha. In contrast to the limited spectrum of oligosaccharide structures present on recombinant bLH(CHO), the endogenous glycoproteins synthesized by CHO cells bear a heterogeneous array of Asn-linked oligosaccharides with 0, 1, 2, 3, or 4 sialic acid moieties. The sialic acid moieties on the Asn-linked oligosaccharides of both endogenous glycoproteins and recombinant bLH(CHO) are exclusively alpha 2,3-linked, suggesting that the alpha 2,6-sialyl-transferase is not active in CHO cells. The bioactivities of bLH(Pit) and bLH(CHO) were compared using MA-10 cells following sequential digestion with neuraminidase and beta-galactosidase. Neither the ED50 (dose producing 50% of the maximum response) for progesterone production (7.2 ng/ml) nor the Pmax (maximum level of progesterone produced) (470 ng/ml) was altered for bLH(Pit) by these treatments, consistent with the absence of either sialic acid or Gal on bLH(Pit). The ED50 for progesterone production by recombinant bLH(CHO) (16.4 ng/ml) was significantly greater than for bLH(Pit) but was reduced to 5.3 ng/ml following removal of terminal sialic acid. Removal of the subterminal Gal was without further effect. The Pmax for bLH(CHO) (180 ng/ml) was not altered by these treatments. The reduction in bLH(CHO) bioactivity caused by the presence of terminal sialic acid suggests that the presence of terminal sulfate on bLH(Pit) oligosaccharides may also reduce its bioactivity and may play a modulatory role in regulating hormone bioactivity.

Highlights

From the $-Department of Pathology, Washington University Medical School, St
In contrast to the limited spectrum of oligosaccharide structures present on recombinant Bovine LH (bLH)(CHO), the endogenous glycoproteins synthesized by CHO cells bear a heterogeneous array of Asn-linked oligosaccharides with 0, 1,2,3, or 4 sialic acid moieties
The reduction in bLH(CH0) bioactivity caused by the presence of terminal sialic acid suggests that the uresence of terminal sulfate on bLH(Pit) olbzosacchaiides may reduce its bioactivity aid may play a modulatory role in regulating hormone bioactivity

Summary

AND METHODS

Metabolic Labeling with ~H]Glucosamine-CHO cells expressing recombinant dimer bLH (LH-20, 10 pM MTX), bovine a-subunit Bound dimer bLH and free bLH0 were eluted with 50 IIIM diethylamine (pH 11.5), and the eluate was immediately neutralized by addition of NaH2P03. This material was concentrated and dialyzed in a Pro-Di-Con The resulting supernatant was brought to 33% saturation with solid ammonium sulfate at 4 “C, and the precipitated proteins were removed by sedimentation at 10,000 X g for 20 min. Buffer salts and released sugars were separated from digested hormone by gel filtration on l-ml columns of Sephadex. Measurement of Hormone Bioactiuity-The bioactivities of native and exoglycosidase-digested bLH(Pit), bLH(CHO), and hCG were determined using MA-10 cells as described (Ascoli, 1981). Statistical analysis of the dose-response curves was performed using the computer-assisted program ALLFIT (DeLean et al, 1978)

RESULTS

16 A N-O 1

DISCUSSION