Characterization of the Microheterogeneity of Recombinant Primate Prolactin: Implications for Posttranslational Modifications of the Hormonein Vivo*

Abstract

Recombinant baboon and monkey prolactins were expressed in murine C127 cells. The hormones were purified from the conditioned media of these cells using a combination of cation, anion, and gel filtration chromatographies. This purification scheme provided approximately a 20-fold purification of the proteins with a 40% cumulative yield. Sodium dodecyl sulfate gel electrophoresis of the purified hormones in conjunction with Coomassie blue staining and immunoblotting procedures revealed three major prolactin-related bands with molecular weights corresponding to Mr 16,000, 23,000, and 27,000. Based on these analyses the samples were judged to be greater than 90% pure. Amino terminal sequence analysis of the purified baboon and monkey hormones provided three distinct prolactin-related sequences for each preparation. The predominant sequence corresponded to the predicted amino terminal sequences of the hormones which began with leucine at position 1. Two minor sequences, individually representing approximately 10-20% of the total population, were also identified; one starting at position 11 and the other at position 133. Carbohydrate compositional analysis of the proteins suggested that greater than 50% of the population were glycosylated with a fucosylated complex oligosaccharide. Analysis of the specific bioactivity of the recombinant hormones in the Nb2 cell proliferation assay showed them to be comparable to the NIH and WHO human pituitary-derived standards.

Roles of prolactin and related members of the prolactin/growth hormone/placental lactogen family in angiogenesis

“…Also, incubation of exogenous PRL with a fibroblast lysate results in the formation of 16K PRL (Corbacho et al 2000b). Finally, a 16K immunoreactive PRL has been detected in eukaryotic cells that express a transfected PRL gene (Cole et al 1991, Yamamoto et al 1992, indicating the existence of proteolytic activity to generate 16K PRL from PRL. Whereas 16K PRL is observed under many physiological/pathophysiological conditions, the identity of the proteolytic enzymes responsible for this posttranslational modification has remained unresolved.…”

“…The possibility of the 14K PRL-like protein being either a proteolytically processed product of 16K PRL or an independent product of PRL proteolysis needs to be addressed. During recombinant synthesis of primate PRL a PRL fragment of approximately 14 kDa that may arise from proteolysis at Ile 133 was observed (Cole et al 1991). The 14K PRL-like protein is localized within the secretory granules of vasopressin-containing cells (Mejía et al 1997) and is released by cultured neurohypophyseal endings (Torner et al 1995).…”

Glycosylated equine prolactin and its carbohydrate moiety

“…A small amount of sialic acid in G-ePRL approximated the galactose content of the preparation. In contrast to recombinant glycosylated prolactins (Cole et al, 1991;Price et al, 1995), the pituitary-derived G-ePRL had a much lower content of Gal and NeuAc. Sulfate analysis of the glycopeptide 29-37 revealed one residue of covalently bound sulfate per molecule, indicating the presence of a sulfated monosaccharide (Fig.…”

“…The same glycosylation sequence is also present in human (Shome and Parlow, 1977), porcine (Li, 1976), equine (Lehrman et al, 1988), whale (Tsubokawa et al, 1985), baboon (Cole et al, 1991), monkey (Cole et al, 1991), camel (Martinat et al, 1991), and mink (Perelygina, 1993) prolactins. G-PRL with an oligosaccharide moiety linked to Asn 31 has been isolated from ovine (Lewis et al, 1984), human (Lewis et al, 1985;Champier et al, 1987;Price et al, 1995), porcine (Pankov and Butnev, 1986;Sinha et al, 1989), baboon (Cole et al, 1991), monkey (Cole et al, 1991), and camel (Martinat et al, 1991) sources. Although the G-PRLs possess the same amino acid sequences as their nonglycosylated counterparts, they show decreased activity in RIA, receptor-binding assays, and bioassays relative to their nonglycosylated forms (Sinha, 1995).…”

“…This decrease in activity is presumably a result of interference by the carbohydrate moiety with the binding of the hormone to the antibodies and receptors involved. Whereas the carbohydrate units of recombinant Asn31-glycosylated prolactins have been well characterized (Cole et al, 1991;Price et al, 1995), reports on the monosaccharide composition and proposed carbohydrate structures of natural pituitary glycosylated prolactins can be quite contradictory (Lewis et al, 1984;Pankov and Butnev, 1986;Butnev et al, 1987;Corcoran and Proudman, 1991;Hoffman et al, 1993).…”