Cell-specific posttranslational processing of the surfactant-associated protein SP-B

1993 ◽  
Vol 264 (3) ◽  
pp. L290-L299 ◽  
Author(s):  
S. Hawgood ◽  
D. Latham ◽  
J. Borchelt ◽  
D. Damm ◽  
T. White ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Specific Protein ◽  
Precursor Protein ◽  
Type Ii Cells ◽  
Posttranslational Processing ◽  
Type Ii ◽  
Clara Cells ◽  
Ovary Cells

Pulmonary surfactant-associated protein B (SP-B) is a 9-kDa lung-specific protein expressed in alveolar epithelial type II cells and Clara cells. The protein markedly increases the surface activity of phospholipids and is an active component in some surfactants in clinical use. SP-B is produced from a 43-kDa precursor protein by proteolytic cleavage of flanking regions from both the NH2- and COOH-terminal ends of the active protein. In this study we have compared the nature of the posttranslational processing of the SP-B precursor in type II cells and in a heterologous cell line transfected with the SP-B precursor. We found that isolated type II cells produce the 9-kDa form of SP-B from the precursor through a series of intermediates detectable in the cell lysates. In contrast Chinese hamster ovary cells stably transfected with the full-length human SP-B precursor produce the precursor and a 26-kDa intermediate but not the 9-kDa protein. The precursor protein in both cell types is glycosylated with NH2-linked sugars. Our results suggest there is cell specificity in the posttranslational processing of the SP-B precursor.

Impaired cell volume regulation in Na(+)-H+ exchange-deficient mutants

1989 ◽  
Vol 257 (6) ◽  
pp. C1158-C1165 ◽  
Author(s):  
D. Rotin ◽  
S. Grinstein
Keyword(s):  
Chinese Hamster Ovary ◽  
Volume Regulation ◽  
Chinese Hamster Ovary Cells ◽  
Cell Volume Regulation ◽  
Chinese Hamster ◽  
Cell Types ◽  
Influx Rate ◽  
Volume Increase ◽  
Ovary Cells ◽  
Na Uptake

To elucidate the mechanism of regulatory volume increase (RVI) in Chinese hamster ovary cells, Na(+)-H+ exchange-deficient mutants, called AP-1, were derived from WT-5 cells, a wildtype subclone. The absence of functional antiports in AP-1 cells was established through measurements of intracellular pH (pHi) and Na+ uptake. Cells exposed to hypotonic medium initially swelled but regained near-normal volume within minutes. When isotonicity was then restored, WT-5 cells shrank immediately and then carried out RVI, which was inhibited by 0.1 mM amiloride. This amiloride-sensitive RVI was absent in the AP-1 mutants, suggesting involvement of Na(+)-H+ exchange. In some cell types, RVI is mediated by Na(+)-K(+)-2Cl- cotransport. Bumetanide-sensitive 86Rb+ (K+) influx was detectable in both WT-5 and AP-1 cells, suggesting the presence of Na(+)-K(+)-2Cl- cotransport. Bumetanide-sensitive influx was stimulated by osmotic shrinking in WT-5 cells, and only slightly in AP-1 cells. However, Na(+)-K(+)-2Cl- cotransport did not contribute to volume regulation, since bumetanide (50 microM) failed to inhibit RVI in osmotically shrunken WT-5 cells. The inability of cotransport to induce a volume gain in WT-5 cells was attributable to the simultaneous stimulation of Na(+)-K(+)-2Cl- efflux. The rate of efflux was similar in magnitude to the corresponding influx rate so that net Na(+)-K(+)-2Cl- cotransport was negligible. These results show that RVI in osmotically shrunken Chinese hamster ovary cells is mediated by the Na(+)-H+ antiport and that, although stimulated, Na(+)-K(+)-2Cl- cotransport does not contribute to anisosmotic volume regulation.

scholarly journals Glycosylation of CD44 negatively regulates its recognition of hyaluronan.

1995 ◽  
Vol 182 (2) ◽  
pp. 419-429 ◽  
Author(s):  
S Katoh ◽  
Z Zheng ◽  
K Oritani ◽  
T Shimozato ◽  
P W Kincade
Keyword(s):  
B Cells ◽  
Ligand Binding ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Characteristics ◽  
Cell Types ◽  
Neuraminidase Treatment ◽  
Ovary Cells ◽  
Transitional State

Although CD44 is expressed on a wide variety of cell types, few of them use it to recognize the ligand hyaluronan (HA). A glycosylation-defective clone of Chinese hamster ovary cells (Lec 8) bound HA, demonstrating that complete processing of glycoproteins with addition of a full complement of sialic acid is not required. On the contrary, subsequent findings revealed that complex sugars on CD44 can actually inhibit ligand recognition. Two subclones of wild-type Chinese hamster ovary cells with similar amounts of surface CD44 were isolated on the basis of HA binding and found to differ with respect to CD44 size as well as staining with fluorescent lectins. Treatment of the nonbinding clone with tunicamycin reduced the size of the protein and allowed the cells to recognize HA via CD44. This function was also induced by treatment with deglycosylating enzymes (either a mixture of endoglycosidase F and N-glycosidase F or neuraminidase alone). A possible role for glycosylation in regulation of adhesion was then sought with a series of normal and transformed murine cells. Disruption of glycosylation or treatment with deglycosylating enzymes did not induce ligand binding in an interleukin 7-dependent pre-B cell line, and splenic B cells also appeared to be in an inactive state. Some normal B cells acquired the ability to recognize HA after stimulation with lipopolysaccharide or interleukin 5 and had distinctive surface characteristics (loss of immunoglobulin D and acquisition of CD43). An additional subset of activated cells might have been in a transitional state, because the cells bound ligand after neuraminidase treatment. The ligand-binding ability of a purified CD44-immunoglobulin fusion protein dramatically increased after neuraminidase treatment. Thus, differential glycosylation of this molecule is sufficient to influence its recognition function. Cell adhesion involving HA can be regulated by multiple mechanisms, one of which involves variable glycosylation of CD44.

Amplification and expression of heterologous ornithine decarboxylase in Chinese hamster cells

1988 ◽  
Vol 8 (2) ◽  
pp. 764-769
Author(s):  
T R Chiang ◽  
L McConlogue
Keyword(s):  
Ornithine Decarboxylase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Wild Type ◽  
Dominant Marker ◽  
Ovary Cells ◽  
Mammalian Expression ◽  
Mammalian Expression Vector

We have developed an amplifiable mammalian expression vector based on the enzyme ornithine decarboxylase (ODC). We show greater than 700-fold amplification of this vector in ODC-deficient Chinese hamster ovary cells. A passive coamplified marker, dihydrofolate reductase (dhfr), was amplified and overexpressed 1,000-fold. This ODC vector was a dominant marker in a variety of cell types and displayed at least 300-fold amplification in wild-type Chinese hamster ovary cells.

scholarly journals Amplification and expression of heterologous ornithine decarboxylase in Chinese hamster cells.

1988 ◽  
Vol 8 (2) ◽  
pp. 764-769 ◽  
Author(s):  
T R Chiang ◽  
L McConlogue
Keyword(s):  
Ornithine Decarboxylase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Wild Type ◽  
Dominant Marker ◽  
Ovary Cells ◽  
Mammalian Expression ◽  
Mammalian Expression Vector

We have developed an amplifiable mammalian expression vector based on the enzyme ornithine decarboxylase (ODC). We show greater than 700-fold amplification of this vector in ODC-deficient Chinese hamster ovary cells. A passive coamplified marker, dihydrofolate reductase (dhfr), was amplified and overexpressed 1,000-fold. This ODC vector was a dominant marker in a variety of cell types and displayed at least 300-fold amplification in wild-type Chinese hamster ovary cells.

scholarly journals BMN 250, a fusion of lysosomal alpha-N-acetylglucosaminidase with IGF2, exhibits different patterns of cellular uptake into critical cell types of Sanfilippo syndrome B disease pathogenesis

2018 ◽  
Author(s):  
Gouri Yogalingam ◽  
Amanda R Luu ◽  
Heather Prill ◽  
Melanie J. Lo ◽  
Bryan Yip ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Sanfilippo Syndrome ◽  
Disease Pathogenesis ◽  
Ovary Cells ◽  
Enzyme Replacement ◽  
Lysosomal Accumulation ◽  
Lysosomal Targeting

ABSTRACTSanfilippo syndrome type B (Sanfilippo B; Mucopolysaccharidosis type IIIB) occurs due to genetic deficiency of lysosomal alpha-N-acetylglucosaminidase (NAGLU) and subsequent lysosomal accumulation of heparan sulfate (HS), which coincides with devastating neurodegenerative disease. Because NAGLU expressed in Chinese hamster ovary cells is not mannose-6-phosphorylated, we developed an insulin-like growth factor 2 (IGF2)-tagged NAGLU molecule (BMN 250; tralesinidase alfa) that binds avidly to the IGF2 / cation-independent mannose 6-phosphate receptor (CI-MPR) for glycosylation independent lysosomal targeting. BMN 250 is currently being developed as an investigational enzyme replacement therapy for Sanfilippo B. Here we distinguish two cellular uptake mechanisms by which BMN 250 is targeted to lysosomes. In normal rodent-derived neurons and astrocytes, the majority of BMN250 uptake over 24 hours reaches saturation, which can be competitively inhibited with IGF2, suggestive of CI-MPR-mediated uptake. Kuptake, defined as the concentration of enzyme at half-maximal uptake, is 5 nM and 3 nM in neurons and astrocytes, with a maximal uptake capacity (Vmax) corresponding to 764 nmol/hr/mg and 5380 nmol/hr/mg, respectively. Similar to neurons and astrocytes, BMN 250 uptake in Sanfilippo B patient fibroblasts is predominantly CI-MPR-mediated, resulting in augmentation of NAGLU activity with doses of enzyme that fall well below the Kuptake (5 nM), which are sufficient to prevent HS accumulation. In contrast, uptake of the untagged recombinant human NAGLU (rhNAGLU) enzyme in neurons, astrocytes and fibroblasts is negligible at the same doses tested. In microglia, receptor-independent uptake, defined as enzyme uptake resistant to competition with excess IGF2, results in appreciable lysosomal delivery of BMN 250 and rhNAGLU (Vmax=12,336 nmol/hr/mg and 5469 nmol/hr/mg, respectively). These results suggest that while receptor-independent mechanisms exist for lysosomal targeting of rhNAGLU in microglia, BMN 250, by its IGF2 tag moiety, confers increased CI-MPR-mediated lysosomal targeting to neurons and astrocytes, two additional critical cell types of Sanfilippo B disease pathogenesis.

Small linear chloride channels are endogenous to nonepithelial cells

1992 ◽  
Vol 263 (3) ◽  
pp. C708-C713 ◽  
Author(s):  
S. E. Gabriel ◽  
E. M. Price ◽  
R. C. Boucher ◽  
M. J. Stutts
Keyword(s):  
Cell Lines ◽  
Chloride Channels ◽  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Ovary Cells ◽  
3T3 Fibroblasts ◽  
Whole Cell Patch Clamp ◽  
Cl Channels

We used both single-channel and whole cell patch-clamp techniques to characterize chloride channels and currents endogenous to Sf9 cells, 3T3 fibroblasts, and Chinese hamster ovary cells. In cell-attached patches from these cell types, anion channels were observed with low ohmic conductance (4-11 ps), linear current-voltage relationships, and little time- or voltage-dependent behavior. These channels are very similar to the Cl- channels reported to appear concomitant with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in these cell lines. The presence of such endogenous channels suggests either that low levels of CFTR are present in all of these cell lines prior to transfection or that an endogenous non-CFTR channel is present in these cell types. Our results suggest that at least some of the channel behaviors attributed to expressed, recombinant CFTR in previous studies may have been due to these endogenous Cl- channels.

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