Asialoglycoprotein receptor mediates the toxic effects of an asialofetuin-diphtheria toxin fragment A conjugate on cultured rat hepatocytes.

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Abstract
A toxic hybrid protein was constructed that is recognized by asialoglycoprotein (ASGP) receptors of cultured rat hepatocytes. The conjugate consists of fragment A of diphtheria (Corynebacterium diphtheriae) toxin (DTA) linked by a disulfide bond to asialofetuin (ASF). This conjugate is highly toxic, inhibitng protein synthesis in primary rat hepatocytes at concentrations as low as 10 pM. The ASF-DTA conjugate was 600 and 1800 times as toxic as diphtheria toxin and DTA, respectively, on primary rat hepatocytes. The ASGP receptor recognizes galactose-terminated proteins. A series of glycoproteins were tested for their ability to block the action of the ASF-DTA conjugate. Fetuin and orosomucoid, 2 glycoproteins with terminal sialic acid on their oligosaccharide chains, did not block the action of the conjugate. Their galactose terminated asialo derivatives, ASF and asialoorosomucoid, as expected, did block the action of the conjugate. The N-acetylglucosaminyl-terminated derivative (asialoagalactoorosomucoid) had no appreciable effect on the activity of the conjugate. The ASF-DTA conjugate was tested on 6 cell types; except for primary rat hepatocytes, none were affected by a high concentration (10 nM) of ASF-DTA conjugate. A fetuin-DTA conjugate was less toxic by a factor of 300 than the ASF-DTA conjugate and exerted its effects primarily through non-receptor-mediated mechanisms. The highly toxic ASF-DTA conjugate is evidently cell-type specific and its action is seemingly mediated by a well-characterized receptor. The mechanism of receptor-ligand internalization were extensively investigated.

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