We have generated a panel of potent, selective monoclonal antibodies that bind human and mouse ␣ v ␤ 6 integrin with high affinity (up to 15 pM). A subset of these antibodies blocked the binding of ␣ v ␤ 6 to the transforming growth factor-␤1 latency-associated peptide with IC 50 values as low as 18 pM, and prevented the subsequent ␣ v ␤ 6 -mediated activation of transforming growth factor-␤1. The antibodies also inhibited ␣ v ␤ 6 binding to fibronectin. The blocking antibodies form two biochemical classes. One class, exemplified by the ligand-mimetic antibody 6.8G6, bound to the integrin in a divalent cation-dependent manner, contained an RGD motif or a related sequence in CDR3 of the heavy chain, was blocked by RGD-containing peptides, and was internalized by ␣ v ␤ 6 -expressing cells. Despite containing an RGD sequence, 6.8G6 was specific for ␣ v ␤ 6 and showed no cross-reactivity with the RGD-binding integrins ␣ v ␤ 3 , ␣ v ␤ 8 , or ␣ IIb ␤ 3 . The nonligand-mimetic blocking antibodies, exemplified by 6.3G9, were cation-independent, were not blocked by RGD-containing peptides, were not internalized, and did not contain RGD or related sequences. These two classes of antibody were unable to bind simultaneously to ␣ v ␤ 6 , suggesting that they may bind overlapping epitopes. The "ligand-mimetic" antibodies are the first to be described for ␣ v ␤ 6 and resemble those described for ␣ IIb ␤ 3 . We also report for the first time the relative abilities of divalent cations to promote ␣ v ␤ 6 binding to latency-associated peptide and to the ligand-mimetic antibodies. These antibodies should provide valuable tools to study the ligand-receptor interactions of ␣ v ␤ 6 as well as the role of ␣ v ␤ 6 in vivo.

The endoplasmic reticulum (ER) protein GT1 (UDP-glucose: glycoprotein glucosyltransferase) is the central enzyme that modifies N-linked carbohydrates based upon the properties of the polypeptide backbone of the maturing substrate. GT1 adds glucose residues to nonglucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate is poorly understood. We analyzed the reglucosylation of a maturing model glycoprotein, influenza hemagglutinin (HA), in the intact mammalian ER. GT1 reglucosylated N-linked glycans in the slow-folding stem domain of HA once the nascent chain was released from the ribosome. Maturation mutants that disrupted the oxidation or oligomerization of HA also supported region-specific reglucosylation by GT1. Therefore, GT1 acts as an ER quality control sensor by posttranslationally reglucosylating glycans on slow-folding or nonnative domains to recruit chaperones specifically to critical aberrant regions.

Eukaryotic secretory pathway cargo fold to their native structures within the confines of the endoplasmic reticulum (ER). To ensure a high degree of folding fidelity, a multitude of covalent and noncovalent constraints are imparted upon nascent proteins. These constraints come in the form of topological restrictions or membrane tethers, covalent modifications, and interactions with a series of molecular chaperones. N-linked glycosylation provides inherent benefits to proper folding, and creates a platform for interactions with specific chaperones and Cys modifying enzymes. Recent insights into this timeline of protein maturation have revealed mechanisms for protein glycosylation, and iterative targeting of incomplete folding intermediates, which provides nurturing interactions with molecular chaperones that assist in the efficient maturation of proteins in the eukaryotic secretory pathway.

The earliest steps in nascent protein maturation greatly affect its overall efficiency. Constraints placed on maturing proteins at these early stages limit available conformations and help to direct the native maturation process. For type II membrane proteins, these cotranslational constraints include N-and C-terminal membrane tethering, chaperone binding, and disulfide bond formation. The cotranslational maturation process for the type II membrane glycoprotein influenza neuraminidase (NA) was investigated to provide a deeper understanding of these initial endoplasmic reticulum events. The type II orientation provides experimental advantages to monitor the first maturation steps. Calnexin was shown to cotranslationally interact with NA prior to calreticulin. These interactions were required for the efficient maturation of NA as it prematurely formed intramolecular disulfides and aggregated when calnexin and calreticulin interactions were abolished. Lectin chaperone binding slowed the NA maturation process, increasing its fidelity. Carbohydrates were required for NA maturation in a regiospecific manner. A subset of NA formed intermolecular disulfides and oligomerized cotranslationally. This fraction increased in the absence of calnexin and calreticulin binding. NA dimerization also occurred for an NA mutant lacking the critical large loop disulfide bond, indicating that dimerization did not require proper NA oxidation. The strict evaluation of proper maturation carried out by the quality control machinery was instilled at the tetramerization step. This study illustrates the type II membrane protein maturation process and shows how important cotranslational events contribute to the proper cellular maturation of glycoproteins.

A natural substrate for UGT1 is confirmed, revealing how the enzyme functions in the calnexin chaperone system as a quality control step in protein folding.